Nope polypeptides, encoding nucleic acids and methods of use

ABSTRACT

The invention provides an isolated Nope polypeptide, or functional fragment thereof, containing the amino acid sequence of a Nope polypeptide (SEQ ID NO:2), or a modification thereof. The invention also provides an isolated nucleic acid molecule encoding a Nope polypeptide amino acid sequence referenced as SEQ ID NO:2, or a modification thereof. The invention additionally provides an isolated nucleic acid molecule containing the nucleotide sequence referenced as SEQ ID NO:1, or a modification thereof. The invention further provides methods of detecting Nope polypeptides and Nope nucleic acid molecules.

[0001] This application claims the benefit of two U.S. ProvisionalApplications No. 60/174,496, filed Jan. 4, 2000, and No. 60/205,789,filed May 19, 2000, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to molecular geneticsand, more specifically, to Nope polypeptides and encoding nucleic acidmolecules.

[0003] All multi-celled organisms develop from a single cell by acomplex process that coordinates the formation of particular tissues,structures and systems in the body to determine the morphology andfunction of the organism. The complex process of development of a singlecell to a complex, multi-celled organism is regulated by the temporaland spatial expression of particular genes.

[0004] In complex, multi-celled organisms, the nervous system provides anetwork that allows the transmission of various signals from outside theorganism to particular organs, tissues or systems, thereby allowing theorganism to respond to external stimuli. The development of the nervoussystem requires the expression of developmentally regulated,tissue-specific genes that encode proteins required for the function ofspecific cell types that form the nervous system.

[0005] The formation of the nervous system in developing embryosrequires the migration of specific types of cells. In the developingcentral nervous system, newly formed neurons migrate along predefinedpathways to establish a variety of distinct structures within the adultbrain. The formation of an axon, the long cellular process of a neuron,involves the navigation of the axon process to specific targets toestablish the intricate networks of the central nervous system.

[0006] Axons function to propagate electrical signals between nervecells and a target, for example, another nerve cell or a target tissue.In order to propagate the electrical signal to the proper target, theaxon of the nerve cell must be guided during development to a particulartarget. During development of the nervous system, the guidance of theaxons to particular targets is mediated by cell surface proteins thatform specific ligand-receptor interactions.

[0007] A family of axon-associated adhesion receptors have beenidentified having a conserved structural motif, specifically animmunoglobulin domain, which resembles a structure found inimmunoglobulins. These adhesion molecules are therefore classified asmembers of an immunoglobulin superfamily. The axon-associated adhesionreceptors function to specifically bind to ligands and mediate cell-cellinteractions in the developing nervous system. Although severalaxon-associated adhesion receptors have been identified, the identity ofall axon guidance receptors that specifically function in guiding axonsto their target and other gene products required for the development ofthe nervous system has not previously been determined.

[0008] Thus, there exists a need to identify genes that regulate thedevelopment of the nervous system and related biological functions. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

[0009] The invention provides an isolated Nope polypeptide, orfunctional fragment thereof, containing the amino acid sequence of aNope polypeptide (SEQ ID NO:2), or a modification thereof. The inventionalso provides an isolated nucleic acid molecule encoding a Nopepolypeptide amino acid sequence referenced as SEQ ID NO:2, or amodification thereof. The invention additionally provides an isolatednucleic acid molecule containing the nucleotide sequence referenced asSEQ ID NO:1, or a modification thereof. The invention further providesmethods of detecting Nope polypeptides and Nope nucleic acid molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows the genomic localization of the Nope gene, thetissue-specific expression of Nope mRNA, and the domain structure ofNope polypeptide.

[0011]FIG. 1A shows the location of expressed sequence tags (ESTs) inthe genomic region upstream of the Punc gene, which are shown as blackbars with the corresponding Genbank accession numbers indicated. Theregion designated e11 is the cloned restriction fragment used togenerate a Nope hybridization probe. The Nope polyadenylation signal andthe ATG start codon of the Punc gene are shown.

[0012]FIG. 1B shows the domain structure of the Nope protein incomparison to Neogenin, DCC, Punc, and NCAM.

[0013]FIG. 2 shows the nucleotide and amino acid sequence of Nope andthe nucleotide sequence of Nope genomic DNA.

[0014]FIG. 2A shows the nucleotide sequence of the Nope cDNA (SEQ IDNO:1).

[0015]FIG. 2B shows the amino acid sequence derived from cDNA clones ofthe Nope gene (SEQ ID NO:2), which is encoded by nucleotides 1-3756 ofFIG. 2a (SEQ ID NO:45). First shaded area corresponds to the signalpeptide (amino acids 1-21); second shaded area corresponds to thetransmembrane domain (amino acids 954-977); the first four underlinedregions correspond to immunoglobulin (Ig) domains (Ig domain 1 (Ig1);amino acids 47-127)(Ig2; amino acids 155-218)(Ig3; amino acids256-318)(Ig4; amino acids 347-411); the last five underlined regionscorrespond to fibronectin-type III (FnIII) domains (FnIII domain 1(Fn1); amino acids 429-511)(Fn2; amino acids 527-609)(Fn3; amino acids630-725)(Fn4; amino acids 750-831)(Fn5; amino acids 848-931).

[0016]FIG. 2C shows the nucleotide sequence of a genomic sequence (SEQID NO:43) encoding the 5′ region of the Nope cDNA. The start codon isshown in bold, the coding region of the first exon (SEQ ID NO:44) isunderlined, and the splice site is shown in italics.

[0017]FIG. 3 shows the evolutionary relationships between Nope and othermembers of the Ig superfamily.

[0018]FIG. 3A shows the evolutionary relationship between Nope and theIg superfamily.

[0019]FIG. 3B shows the evolutionary relationship between individual Igdomains derived from Nope, Punc, DCC, and Neogenin.

[0020]FIG. 3C shows the sequence relationship between Nope and Punc asshown by dot plot analysis based on a PAM similarity matrix. Sequencesimilarities appear as diagonal lines.

[0021]FIG. 4 shows chromosomal mapping of Nope to chromosome 9.Structures of the encoded proteins are indicated next to the chromosomesketch. Placement of Neogenin, Nope, Punc, and BAC end markers relativeto framework markers D9Mit48 and D9Mit143 on chromosome 9 are shown.Distances are given in centiRays (cR). The arrangement of BAC clones andthe origin of PCR products used for mapping is shown on the right.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides Nope polypeptides and encodingnucleic acids. The invention also provides methods for detecting nucleicacids encoding Nope and methods for detecting Nope polypeptides. Themethods of the invention are advantageous for specifically detecting thepresence of a Nope polypeptide or a nucleic acid encoding Nope in asample.

[0023] Nope is a newly identified mouse gene located on chromosome 9. Asdisclosed herein, the Nope polypeptide encoded by the Nope gene containsfour immunoglobulin domains and five fibronectin-type III repeats, asingle transmembrane domain and a cytoplasmic domain. Nope is a newmember of the immunoglobulin superfamily of cell surface proteins andhas a high level of similarity to Punc and to guidance receptors such asDeleted in Colorectal Cancer (DCC) and Neogenin. Nope is expressedduring embryonic development in the notochord, in developing skeletalmuscles, and later in the ventricular zone of the nervous system. In theadult brain, Nope is present in the hippocampus.

[0024] As used herein, the term “functional fragment,” when used inreference to a Nope polypeptide, is intended to refer to a portion of aNope polypeptide that retains some or all or the activity of a Nopepolypeptide. Exemplary functional fragments of a Nope polypeptideinclude the intracellular domain, the extracellular domain, the fourindividual immunoglobulin domains and the five individualfibronectin-type III domains. A functional domain contains an activitythat is recognizable as a Nope polypeptide. For example, anintracellular domain contains the functional activity that mediates thesignaling properties of the Nope polypeptide. The extracellular domaincontains the functional activity of binding to a Nope ligand. Theimmunoglobulin and fibronectin domains are functional motifs thatcontribute to the structure and binding activity of the Nopeextracellular domain.

[0025] In addition, other functional fragments of Nope are recognizableas providing a Nope polypeptide function. For example, a polypeptidefragment of Nope is recognizable as a functional fragment if thefragment can specifically bind to an antibody specific for a Nopepolypeptide. Other functional fragments of a Nope polypeptide includeNope peptide fragments that are functional antigenic fragments, whichcan be used to generate a Nope-specific antibody.

[0026] As used herein, the term “polypeptide” when used in reference toNope is intended to refer to a peptide or polypeptide of two or moreamino acids. A “modification” of a Nope polypeptide can include aconservative substitution of the Nope amino acid sequence. Conservativesubstitutions of encoded amino acids include, for example, amino acidsthat belong within the following groups: (1) non-polar amino acids (Gly,Ala, Val, Leu, and Ile); (2) polar neutral amino acids (Cys, Met, Ser,Thr, Asn, and Gln); (3) polar acidic amino acids (Asp and Glu); (4)polar basic amino acids (Lys, Arg and His); and (5) aromatic amino acids(Phe, Trp, Tyr, and His). Other minor modifications are included withinNope polypeptides so long as the polypeptide retains some or all of itsfunction as described herein.

[0027] A modification of a polypeptide can also include derivatives,analogues and functional mimetics thereof. For example, derivatives caninclude chemical modifications of the polypeptide such as alkylation,acylation, carbamylation, iodination, or any modification thatderivatives the polypeptide. Analogues can include modified amino acids,for example, hydroxyproline or carboxyglutamate, and can include aminoacids that are not linked by peptide bonds. Mimetics encompass chemicalscontaining chemical moieties that mimic the function of the polypeptide.For example, if a polypeptide contains two charged chemical moietieshaving functional activity, a mimetic places two charged chemicalmoieties in a spatial orientation and constrained structure so that thecharged chemical function is maintained in three-dimensional space.Thus, a mimetic, which orients functional groups that provide a functionof Nope, are included within the meaning of a Nope derivative. All ofthese modifications are included within the term “polypeptide” so longas the Nope polypeptide or functional fragment retains its function.

[0028] As used herein, the term “substantially” or “substantially thesame” when used in reference to a nucleotide or amino acid sequence isintended to mean that the nucleotide or amino acid sequence shows aconsiderable degree, amount or extent of sequence identity when comparedto a reference sequence. Such considerable degree, amount or extent ofsequence identity is further considered to be significant and meaningfuland therefore exhibit characteristics which are definitivelyrecognizable or known. A substantially the same amino acid sequenceretains comparable functional and biological activity characteristic ofthe reference polypeptide.

[0029] As used herein, the term “nucleic acid” means a polynucleotidesuch as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) andencompasses both single-stranded and double-stranded nucleic acid aswell as an oligonucleotide. Nucleic acids useful in the inventioninclude genomic DNA, cDNA and mRNA and can represent the sense strand,the anti-sense strand, or both. A genomic sequence of the inventionincludes regulatory regions such as promoters and enhancers thatregulate Nope expression and introns that are outside of the exonsencoding a Nope but does not include proximal genes that do not encodeNope. Exemplary Nope nucleic acids include the nucleotide sequencereferenced as SEQ ID NOS:1 and 43, or fragments thereof. The term“isolated” in reference to a Nope nucleic acid molecule is intended tomean that the molecule is substantially removed or separated fromcomponents with which it is naturally associated, or otherwise modifiedby a human hand, thereby excluding Nope nucleic acid molecules as theyexist in nature.

[0030] As used herein, the term “oligonucleotide” refers to a nucleicacid molecule that includes at least 15 contiguous nucleotides from areference nucleotide sequence, and can include at least 16, 17, 18, 19,20 or at least 25 contiguous nucleotides, and often includes at least30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, up to 350 contiguous nucleotides from the reference nucleotidesequence. The reference nucleotide sequence can be the sense strand orthe anti-sense strand. The oligonucleotide can be chemically synthesizedor expressed recombinantly.

[0031] As used herein, a “modification” of a nucleic acid can alsoinclude one or several nucleotide additions, deletions, or substitutionswith respect to a reference sequence. A modification of a nucleic acidcan include substitutions that do not change the encoded amino acidsequence due to the degeneracy of the genetic code. Such modificationscan correspond to variations that are made deliberately, or which occuras mutations during nucleic acid replication. As such, a modification ofa nucleic acid includes a substantially the same sequence, which isrecognizable as a parent nucleic acid molecule such as the Nopenucleotide sequence referenced as SEQ ID NO:1. For example, asubstantially the same nucleotide sequence can hybridize to thereference nucleotide sequence under moderately stringent or higherstringency conditions.

[0032] Exemplary modifications of the recited Nope sequences includesequences that correspond to homologs of other species such as human,primates, rat, rabbit, bovine, porcine, ovine, canine, feline or otheranimal species. The sequences of corresponding Nopes of non-mousespecies can be determined by methods known in the art, such as by PCR orby screening genomic, cDNA or expression libraries.

[0033] Another exemplary modification of the recited Nope can correspondto splice variant forms of the Nope nucleotide sequence. Additionally, amodification of a nucleotide sequence can include one or more non-nativenucleotides, having, for example, modifications to the base, the sugar,or the phosphate portion, or having a modified phosphodiester linkage.Such modifications can be advantageous in increasing the stability ofthe nucleic acid molecule.

[0034] Furthermore, a modification of a nucleotide sequence can include,for example, a detectable moiety, such as a radiolabel, a fluorochrome,a ferromagnetic substance, a luminescent tag or a detectable bindingagent such as biotin. Such modifications can be advantageous inapplications where detection of a Nope nucleic acid molecule is desired.

[0035] As used herein, a “vector” refers to a recombinant DNA or RNAplasmid or virus that comprises a polynucleotide. A vector can includean expression element operationally linked to a polynucleotide such thatthe expression element controls the expression of the polynucleotide. An“expression element” is a nucleotide sequence involved in an interactionof molecules that contributes to the functional regulation of apolynucleotide, including replication, transcription, splicing,translation, or degradation of the polynucleotide. An expression elementthat controls transcription of a gene can be a promoter, the site ofinitiation of transcription, or an enhancer, a DNA sequence thatincreases the rate of transcription.

[0036] As used herein, the term “sample” is intended to mean anybiological fluid, cell, tissue, organ or portion thereof, that includesor potentially includes Nope nucleic acids or polypeptides. The termincludes samples present in an individual as well as samples obtained orderived from the individual. For example, a sample can be a histologicsection of a specimen obtained by biopsy, or cells that are placed in oradapted to tissue culture. A sample further can be a subcellularfraction or extract, or a crude or substantially pure nucleic acid orprotein preparation. A sample can also be chemically synthesized, forexample, by synthesizing degenerate oligonucleotides.

[0037] As used herein, the term “specifically hybridize” refers to theability of a nucleic acid molecule to hybridize, under at leastmoderately stringent conditions or higher stringency conditions, asdescribed herein, to a reference Nope nucleic acid molecule, withouthybridization under the same conditions with nucleic acid molecules thatare not Nope nucleic acid molecules, such as actin cDNA. Therefore, anucleic acid molecule that specifically hybridizes to a Nope nucleicacid under high stringency conditions would not hybridize to a non-Nopenucleic acid under high stringency conditions.

[0038] The invention provides an isolated Nope polypeptide, orfunctional fragment thereof, comprising the amino acid sequence of aNope polypeptide (SEQ ID NO:2), or a modification thereof. As disclosedherein, Nope was identified as a new member of the immunoglobulinsuperfamily that includes DCC, Neogenin and Punc.

[0039] Proteins of the immunoglobulin superfamily play essential rolesin many biological functions of the surface of cells. This proteinfamily is characterized by the presence of immunoglobulin (Ig) domainsin the extracellular protein moiety and includes cell surface receptorsfor diffusible ligands as well as proteins that mediate cell adhesion(Brummendorf and Rathjen, Curr. Opin. Neurobiol. 6:584-593 (1996)).These proteins in general can act as signal transduction devices thatcan couple the presence of an extracellular cue to second messengerpathways inside the cell. While receptor tyrosine kinases andphosphatases can exert their influence on intracellular signalingdirectly (Holland et al., Curr. Opin. Neurobiol. 8:117-127 (1998)),there is evidence that classical cell adhesion molecules, which wereoriginally thought to only provide mechanochemical linkage (Edelman andCrossin, Annu. Rev. Biochem. 60:155-190 (1991)), can associate withintracellular kinases (Maness et al., Perspect. Dev. Neurobiol.4:169-181 (1996)) and thereby signal, for example, the presence of afavorable environment for cell migration. Many members of this proteinfamily therefore play important roles in tissue formation andmorphogenesis during embryonic development.

[0040] A subgroup of the Ig superfamily has been associated withmigration and guidance of axonal growth cones during development of thevertebrate nervous system and is represented by the protein Deleted inColorectal Cancer (DCC). Originally identified as a tumor suppressor(Fearon et al., Science 247:49-56 (1990)), DCC is a receptor for theaxonal guidance cue Netrin-1 (Keino-Masu et al., Cell 87:175-285 (1996);Kennedy et al., Cell 78:425-435 (1994)). Members of this Ig superfamilysubgroup are type I transmembrane proteins, with four Ig domains intheir extracellular domain, and include frazzled in Drosophila(Kolodziej et al., Cell 87:197-204 (1996)), UNC-40 in C. elegans (Chanet al., Cell 87:187-195 (1996)), and Neogenin in vertebrates (Meyerhardtet al., Oncogene 14:1129-1136 (1997); Vielmetter et al., Genomics41:414-421 (1997)).

[0041] DCC is characterized by the presence of four Ig domains in theextracellular domain. Other members of this Ig superfamily are presentin invertebrate species, where frazzled and UNC-40 represent the DCChomologue in Drosophila and C. elegans, respectively. In vertebrates,DCC functions in the guidance of axonal growth cones and constitutespart of the receptor for the guidance cue Netrin-1 (Keino-Masu et al.,supra, 1996; Kennedy et al., supra, 1994). Frazzled and UNC-40 perform asimilar function in the respective invertebrate species (Chan et al.,supra, 1996; Kolodziej et al., supra, 1996). Discovery of the gene forNeogenin (Meyerhardt et al., supra, 1997; Vielmetter et al., supra,1997) revealed the existence of a second vertebrate homologue that caninteract with Netrin-1 as well (Keino-Masu et al., supra, 1996). Whilethe function of DCC in axon guidance has been confirmed in vitro (de laTorre et al., Neuron 19:1211-1124 (1997); Keino-Masu et al., supra,1996) and in vivo (Fazeli et al., Nature 386:796-804 (1997)), thefunction of Neogenin is less clear, with implications that it canfunction as a guidance receptor for cell migration (Gad et al., supra,1997; Keeling et al., Oncogene 15:691-700 (1997)).

[0042] A more distant member of the Ig superfamily subgroup thatincludes DCC is the mouse protein Punc (Salbaum, Mech. Dev. 71:201-204(1998)). Punc was identified in a screen for genes regulated by thehomeodomain transcription factor Is1-1 (Salbaum, supra, 1998; Karlssonet al., Nature 344:879-882 (1990)), which is essential for motor neurondevelopment (Pfaff et al., Cell 84:309-320 (1996)). Punc has four Igdomains, as with other members of this family, but is a smaller proteinwith only two fibronectin-type III repeats in the extracellular domain,in contrast to six in other members of this family. Punc also differs inits regulation from other vertebrate members of this family. Both DCCand Neogenin have their onset of expression around mid-gestation inmice, increase their expression level, and expand in their expressiondomain during development (Gad et al., Dev. Biol. 192:258-273 (1997)).In contrast, Punc is expressed early after gastrulation in mouse embryosbut undergoes a sharp down regulation after 11 days of gestation(Salbaum, supra, 1998). Expression of Punc is correlated with regions ofproliferating cells, whereas DCC and Neogenin expression is in generalassociated with cells that have started to differentiate.

[0043] The down regulation of Punc is first evident in motor neurons ofthe spinal cord and constitutes an early step of motor neurondifferentiation. Therefore, regulation of Punc and other cell surfacereceptors such as DCC and Neogenin demonstrates the role oftranscriptional control in regulating cell surface properties, which cancontribute to development and cell differentiation. To investigate theregulatory mechanism that controls the expression of the Punc gene, thegenomic region encompassing the Punc gene and its upstream region wascloned (Salbaum, Genome 10:107-111 (1999)).

[0044] As disclosed herein, another gene was found located very close tothe Punc gene, with a polyadenylation site not more that 3.5 kb from theATG start codon of Punc (Example I). The newly identified gene close toPunc is a novel member of the immunoglobulin superfamily and, similar toPunc, belongs to the DCC subgroup. This newly identified gene is calledNope, for Neighbor of Punc ell, where ell is the probe used to identifythe gene (see Example I).

[0045] Cloning genes of more distant members of the Ig subgroup such asPunc (Salbaum, supra, 1998) and Nope, as disclosed herein, suggestedthat the DCC Ig superfamily subgroup is more diverse than originallyappreciated, and the subgroup can be referred to as the DEAL family orsubgroup, for DCC et al. This diversity likely reflects recent events inthe evolutionary history of vertebrates, since neither Punc nor Nopehave a clear homologue in the nematode C. elegans (see Example III). Theonly protein sequence derived from the C. elegans genome that displaysthe characteristic four Ig domains is UNC-40, which is the homologue ofDCC (Chan et al., supra, 1996). The presence of more than one gene ofthe DEAL subgroup is likely a recent acquisition in vertebrates.

[0046] The common structural motif of the DEAL proteins is the presenceof four Ig domains, with the highest degree of similarity located in thefourth, innermost domain. It is likely that this domain is essential forextracellular interactions, in particular the binding of Netrin-1 to DCCor Neogenin (Gad et al., supra, 1997). All Ig domains display sequencefeatures that classifies them as V type domains (Vaughn and Bjorkman,Neuron 16:261-273 (1996)). In contrast to the conserved number andsequence of the Ig domains, the number of FnIII repeats can vary and isdiverse in the more distant members Punc and Nope (see Example III).Core members of the DEAL subgroup, for example, DCC, Neogenin, frazzled,and UNC-40, all have six FnIII repeats, whereas Nope has five and Punchas only two FnIII repeats. In addition to variation in the overallFnIII domain configuration, there is significant diversity in amino acidsequences. Together, the degree of conservation observed with thesequence and domain configuration suggests that the Ig domains are underhigher selective pressure than the FnIII repeats. This supports the viewthat in DEAL proteins, as in other Ig CAMs (Brummendorf and Rathjen,supra, 1996), the biological interactions are executed via the Igdomain(s), whereas the FnIII repeats provide a structural function.

[0047] The cytoplasmic domains of Nope and Punc are also substantiallydistinct form core members of the DEAL family (see Example III). Boththe Nope and the Punc sequence display no structural similarity to eachother, to other cytoplasmic domains of the DEAL family, or to otherprotein domains or motifs in protein sequence databases. It has beendemonstrated that DCC is engaged in multiple pathways of signaltransduction, from interfacing with cAMP-dependent second messengercascades during Netrin-1-dependent steering of axonal growth cones (Minget al., Neuron 19:1225-1235 (1997)) to induction of apoptosis in theabsence of the Netrin-1 ligand (Mehlen et al., Nature 395:801-804(1998)). The structural correlate for these functions is thought toreside in the cytoplasmic domain. Due to the sequence divergence, it islikely that Nope-dependent signaling, for example, during myocytedifferentiation, occurs through other proteins or pathways.

[0048] The invention provides an isolated Nope polypeptide, orfunctional fragment thereof. The isolated Nope polypeptides and peptidesof the invention can be prepared by methods known in the art, includingbiochemical, recombinant and synthetic methods. For example, a Nopepolypeptide can be purified by routine biochemical methods from a cellor tissue source that expresses the corresponding Nope transcript orpolypeptide. The methods disclosed herein can be adapted for determiningwhich cells and tissues, and which subcellular fractions therefrom, areappropriate starting materials. Biochemical purification can include,for example, steps such as solubilization of the appropriate tissue orcells, isolation of desired subcellular fractions, size, ion exchange oraffinity chromatography, electrophoresis, and immunoaffinity procedures.The methods and conditions for biochemical purification of a polypeptideof the invention can be chosen by those skilled in the art, andpurification monitored, for example, by an immunological assay or afunctional assay.

[0049] The invention also provides a functional fragment of a Nopepolypeptide. A functional fragment of a Nope polypeptide can be, forexample, the extracellular domain of a Nope polypeptide, correspondingto amino acids 22-953 (SEQ ID NO:4). Additionally, a functional fragmentcan be the intracellular domain of a Nope polypeptide corresponding toamino acids 978-1252 (SEQ ID NO:6). The invention further provides aNope polypeptide functional fragment comprising the amino acid sequenceof immunoglobulin domain 1 (Ig1; amino acids 47-127; SEQ ID NO:8);immunoglobulin domain 2 (Ig2; amino acids 155-218; SEQ ID NO:10);immunoglobulin domain 3 (Ig3; amino acids 256-318; SEQ ID NO:12);immunoglobulin domain 4 (Ig4; amino acids 347-411; SEQ ID NO:14);fibronectin-type III domain 1 (Fn1; amino acids 429-511; SEQ ID NO:16);fibronectin-type III domain 2 (Fn2; amino acids 527-609; SEQ ID NO:18);fibronectin-type III domain 3 (Fn3; amino acids 630-725; SEQ ID NO:20);fibronectin-type III domain 4 (Fn4; amino acids 750-831; SEQ ID NO:22);or fibronectin-type III domain 5 (Fn5; amino acids 848-931; SEQ IDNO:24).

[0050] The invention also provides antibodies that specifically bind aNope polypeptide. As used herein, the term “antibody” is used in itsbroadest sense to include polyclonal and monoclonal antibodies, as wellas antigen binding fragments of such antibodies. With regard to ananti-Nope antibody of the invention, the term “antigen” means a nativeor synthesized Nope polypeptide or fragment thereof.

[0051] An anti-Nope antibody, or antigen binding fragment of such anantibody, is characterized by having specific binding activity for aNope polypeptide or a peptide portion thereof of at least about 1×10⁵M⁻¹. Thus, Fab, F(ab′)₂, Fd and Fv fragments of an anti-Nope antibody,which retain specific binding activity for a Nope polypeptide, areincluded within the definition of an antibody. Specific binding activityof a Nope polypeptide can be readily determined by one skilled in theart, for example, by comparing the binding activity of an anti-Nopeantibody to a Nope polypeptide versus a control polypeptide that is nota Nope polypeptide. Methods of preparing polyclonal or monoclonalantibodies are well known to those skilled in the art (see, for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988)). When using polyclonal antibodies, thepolyclonal sera can be affinity purified using the antigen to generatemono-specific antibodies having reduced background binding and a higherproportion of antigen-specific antibodies.

[0052] In addition, the term “antibody” as used herein includesnaturally occurring antibodies as well as non-naturally occurringantibodies, including, for example, single chain antibodies, chimeric,bifunctional and humanized antibodies, as well as antigen-bindingfragments thereof. Such non-naturally occurring antibodies can beconstructed using solid phase peptide synthesis, can be producedrecombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al. (Science 246:1275-1281 (1989)).These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art (Winter and Harris, Immunol. Today 14:243-246(1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra,1988); Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford UniversityPress 1995)).

[0053] Anti-Nope antibodies can be raised using a Nope immunogen such asan isolated Nope polypeptide having the amino acid sequence of SEQ IDNO:2, or a fragment thereof, which can be prepared from natural sourcesor produced recombinantly, or a peptide portion of the Nope polypeptide.Such peptide portions of a Nope polypeptide are functional antigenicfragments if the antigenic peptides can be used to generate aNope-specific antibody. A non-immunogenic or weakly immunogenic Nopepolypeptide or portion thereof can be made immunogenic by coupling thehapten to a carrier molecule such as bovine serum albumin (BSA) orkeyhole limpet hemocyanin (KLH). Various other carrier molecules andmethods for coupling a hapten to a carrier molecule are well known inthe art (see, for example, Harlow and Lane, supra, 1988). An immunogenicNope polypeptide fragment can also be generated by expressing thepeptide portion as a fusion protein, for example, to glutathione Stransferase (GST), polyHis or the like. Methods for expressing peptidefusions are well known to those skilled in the art (Ausubel et al.,Current Protocols in Molecular Biology (Supplement 47), John Wiley &Sons, New York (1999)).

[0054] The invention also provides a method of detecting a Nopepolypeptide by contacting a sample with an antibody that specificallybinds a Nope polypeptide and detecting specific binding of the antibody.An anti-Nope antibody is therefore useful, for example, for determiningthe presence or level of a Nope polypeptide in a sample.

[0055] An anti-Nope antibody is also useful for cloning a nucleic acidmolecule encoding a gene encoding a polypeptide immunologically relatedto a Nope polypeptide from an appropriate expression library, forexample, a lambda gt11 library. An anti-Nope antibody also can be usedto substantially purify Nope from a sample, for example, from a cellextract of a cell or tissue expressing Nope or a cell extract from acell expressing a Nope polypeptide from a recombinant nucleic acidmolecule.

[0056] The invention also provides methods for detecting a Nopepolypeptide in a sample by contacting the sample with an agent specificfor Nope under conditions that allow specific binding of the agent to aNope polypeptide and detecting the specifically bound agent. An agentspecific for Nope is a molecule that specifically binds Nope. An exampleof a molecule that specifically binds Nope is a Nope antibody, orantigen binding fragment thereof. Additionally, the Nope binding andmodulatory compounds identified in screening methods, as describedbelow, are also suitable agents that can be used in methods of detectingNope polypeptides.

[0057] Assays for detecting Nope polypeptides include, for example,immunohistochemistry, immunofluorescence, ELISA assays,radioimmunoassay, FACS analysis, immunoprecipitation, immunoblotanalysis, and flow cytometry, using antibodies or antigen bindingfragments specific for Nope (Harlow and Lane, supra, 1988; Harlow andLane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press(1999)). Various immunoassays are well known in the art, and can bereadily modified by those skilled in the art in cases in which the agentis a Nope binding molecule other than an antibody. If desired, the agentor antibody can be rendered detectable by incorporation of, or byconjugation to, a detectable moiety, or binding to a secondary moleculethat is itself detectable or detectably labeled.

[0058] A Nope polypeptide or an anti-Nope antibody can be labeled so asto be detectable using methods well known in the art (Hermanson,Bioconjugate Techniques, Academic Press, 1996; Harlow and Lane, supra,1988). For example, the peptide or antibody can be labeled with variousdetectable moieties including a radiolabel, an enzyme, biotin or afluorochrome. Reagents for labeling a peptide or antibody can beincluded in a kit containing the peptide or antibody or can be purchasedseparately from a commercial source. The invention further provides akit, which contains a Nope polypeptide or an anti-Nope antibody or both.Such a kit also can contain a reaction cocktail that provides the properconditions for performing an assay, for example, an ELISA or otherimmunoassay for determining the level of expression of a Nopepolypeptide in a sample, and can contain control samples that containknown amounts of a Nope polypeptide and, if desired, a second antibodyspecific for the anti-Nope antibody. Where the kit is to be used for animmunoassay, it can include a simple method for detecting the presenceor amount of a Nope polypeptide in a sample that is bound to theantibody.

[0059] The invention also provides an isolated nucleic acid moleculeencoding a Nope polypeptide amino acid sequence referenced as SEQ IDNO:2, or a modification thereof. Such a nucleic acid molecule includesdegenerate nucleotide sequences that encode the amino acid sequencereferenced as SEQ ID NO:2. Additionally, the invention provides anisolated Nope nucleic acid molecule comprising the nucleotide sequencereferenced as SEQ ID NO:1, or a modification thereof.

[0060] The invention additionally provides nucleic acid molecules havingnucleotide sequences that encode functional fragments of a Nopepolypeptide. For example, the invention provides a nucleotide sequenceencoding the extracellular domain of a Nope polypeptide, correspondingto nucleotides 64-2859 (SEQ ID NO:3). Additionally, the inventionprovides a nucleotide sequence encoding the intracellular domain of aNope polypeptide, corresponding to nucleotides 2932-3756 (SEQ ID NO:5).The invention further provides a nucleotide sequence encodingimmunoglobulin domain 1 (Ig1; nucleotides 139-381; SEQ ID NO:7);immunoglobulin domain 2 (Ig2; nucleotides 463-654; SEQ ID NO:9);immunoglobulin domain 3 (Ig3; nucleotides 766-954; SEQ ID NO:11);immunoglobulin domain 4 (Ig4; nucleotides 1039-1233; SEQ ID NO:13);fibronectin-type III domain 1 (Fn1; nucleotids 1285-1533; SEQ ID NO:15);fibronectin-type III domain 2 (Fn2; nucleotides 1579-1827; SEQ IDNO:17); fibronectin-type III domain 3 (Fn3; nucleotides 1888-2175; SEQID NO:19); fibronectin-type III domain 4 (Fn4; nucleotides 2248-2493;SEQ ID NO:21); or fibronectin-type III domain 5 (Fn5; nucleotides2542-2793; SEQ ID NO:23).

[0061] The invention also provides a modification of a Nope nucleotidesequence that hybridizes to a Nope nucleic acid molecule, for example, anucleic acid molecule referenced as SEQ ID NO:1, under at leastmoderately stringent conditions. Modifications of Nope nucleotidesequences, where the modification has at least 60% identity to a Nopenucleotide sequence, are also provided. The invention also providesmodification of a Nope nucleotide sequence having at least 65% identity,at least 70% identity, at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity, or at least 95% identity to aNope nucleic acid such as that referenced as SEQ ID NO:1.

[0062] Moderately stringent conditions, as used herein, refers tohybridization conditions that permit a nucleic acid molecule to bind anucleic acid that has substantial identity to a reference sequence.Moderately stringent conditions include conditions equivalent tohybridization of filter-bound nucleic acid in 50% formamide, 5×Denhart's solution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in0.2× SSPE, 0.2% SDS, at 42° C. In contrast, “highly stringentconditions” include conditions equivalent to hybridization offilter-bound nucleic acid in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2× SSPE, 0.2% SDS, at65° C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone,and 1% bovine serum albumin (BSA). 20× SSPE (sodium chloride, sodiumphosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodiumchloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitablemoderately stringent and highly stringent hybridization buffers andconditions, including varying salt and temperature conditions, are wellknown to those of skill in the art and are described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, Plainview, New York (1989); and Ausubel et al.,supra, 1999).

[0063] In general, a nucleic acid molecule that hybridizes to a recitedsequence under moderately stringent conditions will have greater thanabout 60% identity, such as greater than about 70% identity or greaterthan about 80% identity to the reference sequence over the length of thetwo sequences being compared. A nucleic acid molecule that hybridizes toa recited sequence under highly stringent conditions will generally havegreater than about 90% identity, including greater than about 95%identity, to the reference sequence over the length of the two sequencesbeing compared. Identity of any two nucleic acid sequences can bedetermined by those skilled in the art based, for example, on a BLAST2.0 computer alignment, using default parameters. BLAST 2.0 searching isavailable at http://www.ncbi.nlm.nih.gov/gorf/b12.html., as described byTatiana et al., FEMS Microbiol Lett. 174:247-250 (1999).

[0064] The isolated Nope nucleic acid molecules of the invention can beused in a variety of diagnostic and therapeutic applications. Forexample, the isolated Nope nucleic acid molecules of the invention canbe used as probes, as described above; as templates for the recombinantexpression of Nope polypeptides; or in screening assays such astwo-hybrid assays to identify cellular molecules that bind Nope.

[0065] The invention also provides isolated Nope oligonucleotidescontaining at least 15 contiguous nucleotides of the Nope nucleotidesequence referenced as SEQ ID NO:1, or the antisense strand thereof.

[0066] The Nope oligonucleotides of the invention that contain at least15 contiguous nucleotides of a reference Nope nucleotide sequence areable to hybridize to Nope under moderately stringent or higherstringency hybridization conditions and thus can be advantageously used,for example, as probes to detect Nope DNA or RNA in a sample, and todetect splice variants thereof; as sequencing or PCR primers; asantisense reagents to block transcription of Nope RNA in cells; or inother applications known to those skilled in the art in whichhybridization to a Nope nucleic acid molecule is desirable.

[0067] It is understood that a Nope nucleic acid molecule, as usedherein, specifically excludes previously known nucleic acid moleculesconsisting of nucleotide sequences having identity with the Nopenucleotide sequence (SEQ ID NO:1), such as Expressed Sequence Tags(ESTs), Sequence Tagged Sites (STSs) and genomic fragments, deposited inpublic databases such as the nr, dbest, dbsts, gss and htgs databases,which are available for searching athttp://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=0, using the programBLASTN 2.0.9 described by Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

[0068] In particular, a Nope nucleic acid molecule specifically excludesnucleic acid molecules consisting of any of the nucleotide sequenceshaving the Genbank (gb), EMBL (emb) or DDBJ (dbj) Accession numbers setforth below: AW049847; AA051759; AA944556; AI154094; AI849335; AI599639;AA177505; AA403350; AA859434; AI429536; W33247; AA942729; AA389134;AV015396; AI046835; AW045411; AV047477; AA942730; AV016480; W83755;AL119290; AA253306; AI368698; D61677 (HUM430B04B); AI693740; AI101752;AA792362; AI339313; N53517; R24357; AV148364; AI653753; AA385851;AA947283; AI741225; AI599551; AI393663; T95325; AA706095; R69087;N53427; T89389; AA252900; R69201; AA086299; F09441 (HSC31F032); R36958;R36959; AA331887; R15778; AC021040 and AW109921.

[0069] The Nope nucleic acid molecules and oligonucleotides of theinvention can be produced or isolated by methods known in the art (see,for example, Sambrook et al., supra, 1989; Ausubel et al., supra, 1999).The method chosen will depend, for example, on the type of nucleic acidmolecule desired. Those skilled in the art, based on knowledge of thenucleotide sequences disclosed herein, can readily isolate Nope nucleicacid molecules as genomic DNA, or desired introns, exons or regulatorysequences therefrom; as full-length cDNA or desired fragments therefrom;or as full-length mRNA or desired fragments therefrom, by methods knownin the art.

[0070] One useful method for producing a Nope nucleic acid molecule ofthe invention involves amplification of the nucleic acid molecule usingPCR and Nope oligonucleotides. Either PCR or RT-PCR can be used toproduce a Nope nucleic acid molecule having any desired nucleotideboundaries. Desired modifications to the nucleic acid sequence can alsobe introduced by choosing an appropriate oligonucleotide primer with oneor more additions, deletions or substitutions. Such nucleic acidmolecules can be amplified exponentially starting from as little as asingle gene or mRNA copy, from any cell, tissue or species of interest.

[0071] The invention additionally provides a method of detecting a Nopenucleic acid molecule in a sample by contacting the sample with a Nopeoligonucleotide under conditions allowing specific hybridization to aNope nucleic acid molecule, and detecting specific hybridization. Alsoprovided are methods for detecting a Nope nucleic acid molecule in asample. The method consists of contacting the sample with a Nope nucleicacid molecule under conditions that allow specific hybridization to aNope nucleic acid and detecting specific hybridization. The Nope nucleicacid molecule can be, for example, a Nope nucleotide sequence referencedas SEQ ID NO:1 or a Nope oligonucleotide containing at least 15contiguous nucleotides of a reference Nope nucleotide sequence such asSEQ ID NO:1.

[0072] The invention additionally provides a method of detecting a Nopenucleic acid molecule in a sample by contacting the sample with two ormore Nope oligonucleotides, amplifying a nucleic acid molecule, anddetecting the amplification. The methods of detecting Nope nucleic acidin a sample can be either qualitative or quantitative, as desired. Forexample, the presence, abundance, integrity or structure of a Nopenucleic acid can be determined, as desired, depending on the assayformat and the probe or primer pair chosen.

[0073] Useful assays for detecting a Nope nucleic acid based on specifichybridization with an isolated Nope nucleic acid molecule are well knownin the art and include, for example, in situ hybridization, which can beused to detect altered chromosomal location of the nucleic acidmolecule, altered gene copy number, and RNA abundance, depending on theassay format used. Other hybridization assays include, for example,Northern blots and RNase protection assays, which can be used todetermine the abundance and integrity of different RNA splice variants,and Southern blots, which can be used to determine the copy number andintegrity of DNA. A Nope hybridization probe can be labeled with anysuitable detectable moiety, such as a radioisotope, fluorochrome,chemiluminescent marker, biotin, or other detectable moiety known in theart that is detectable by analytical methods.

[0074] Useful assays for detecting a Nope nucleic acid in a sample basedon amplifying a Nope nucleic acid with two or more Nope oligonucleotidesare also well known in the art, and include, for example, qualitative orquantitative polymerase chain reaction (PCR); reverse-transcription PCR(RT-PCR); single strand conformational polymorphism (SSCP) analysis,which can readily identify a single point mutation in DNA based ondifferences in the secondary structure of single-strand DNA that producean altered electrophoretic mobility upon non-denaturing gelelectrophoresis; and coupled PCR, transcription and translation assays,such as a protein truncation test, in which a mutation in DNA isdetermined by an altered protein product on an electrophoresis gel.Additionally, the amplified Nope nucleic acid can be sequenced to detectmutations and mutational hot-spots, and specific assays for large-scalescreening of samples to identify such mutations can be developed.

[0075] The invention further provides a kit containing a Nope nucleicacid molecule, for example, a Nope nucleotide sequence referenced as SEQID NO:1 or a Nope oligonucleotide of the invention. For example, thediagnostic nucleic acids can be derived from any portion of SEQ ID NO:1or an anti-sense strand thereof. Kits of the invention are useful asdiagnostic systems for assaying for the presence or absence of nucleicacid encoding Nope in either genomic DNA, mRNA or cDNA.

[0076] A suitable diagnostic system includes at least one inventionnucleic acid and can contain two or more invention nucleic acids as aseparately packaged chemical reagent(s) in an amount sufficient for atleast one assay. Instructions for use of the packaged reagent are alsotypically included. Those of skill in the art can readily incorporateinvention nucleic acid probes and/or oligonucleotides useful as primersinto kit form in combination with appropriate buffers and solutions forthe practice of the invention methods as described herein.

[0077] The Nope nucleic acid molecules of the invention can be used toscreen for nucleic acid molecules related to a Nope gene. Nucleic acidmolecules related to Nope can be identified, for example, by screening alibrary, such as a genomic library, cDNA library or expression library,with a detectable agent. Such libraries are commercially available orcan be produced from any desired tissue, cell, or species of interestusing methods known in the art. For example, a cDNA or genomic librarycan be screened by hybridization with a detectably labeled nucleic acidmolecule having a nucleotide sequence disclosed herein. Additionally, anexpression library can be screened with an antibody raised against apolypeptide corresponding to the coding sequence of a Nope nucleic aciddisclosed herein. The library clones containing Nope molecules of theinvention can be isolated from other clones by methods known in the artand, if desired, fragments therefrom can be isolated by restrictionenzyme digestion and gel electrophoresis.

[0078] The invention also provides a vector containing a Nope nucleicacid molecule. The vectors of the invention are useful for subcloningand amplifying a Nope nucleic acid molecule and for recombinantlyexpressing a Nope polypeptide. A vector of the invention can include,for example, viral vectors such as a bacteriophage, a baculovirus or aretrovirus; cosmids or plasmids; and, particularly for cloning largenucleic acid molecules, bacterial artificial chromosome vectors (BACs)and yeast artificial chromosome vectors (YACs). Such vectors arecommercially available, and their uses are well known in the art.

[0079] The invention additionally provides a host cell containing avector comprising a Nope nucleic acid molecule. Exemplary host cellsthat can be used to express recombinant Nope molecules include mammalianprimary cells; established mammalian cell lines, such as COS, CHO, HeLa,NIH3T3, HEK 293 and PC12 cells; amphibian cells, such as Xenopus embryosand oocytes; and other vertebrate cells. Exemplary host cells alsoinclude insect cells such as Drosophila, yeast cells such asSaccharomyces cerevisiae, Saccharomyces pombe, or Pichia pastoris, andprokaryotic cells such as Escherichia coli.

[0080] The invention also provides methods of identifying cellular andnon-cellular molecules that modulate Nope expression and activity. Thesemolecules can be used, for example, in therapeutic applications topromote or inhibit a biological function of Nope.

[0081] As disclosed herein, the intracellular domain of the Nopepolypeptide of the invention functions to mediate intracellularsignaling. By specifically binding particular cellular proteins, theintracellular domain contributes to the function of Nope polypeptide,for example, in axonal guidance or proliferation of developing neurons.Such cellular proteins are themselves likely to have positive ornegative effects on Nope activity, and are also appropriate targets fortherapeutic intervention to prevent or treat disorders associated withaberrant Nope expression. Furthermore, peptides or analogs correspondingto the Nope binding interface of such cellular proteins, or of Nope, canbe administered as therapeutic compounds to specifically interfere withNope function.

[0082] Various binding assays to identify cellular proteins thatinteract with protein binding domains are known in the art and include,for example, yeast two-hybrid screening assays (see, for example, U.S.Pat. Nos. 5,283,173, 5,468,614 and 5,667,973; Ausubel et al., supra,1999; Luban et al., Curr. Opin. Biotechnol. 6:59-64 (1995)) and affinitycolumn chromatography methods using cellular extracts. By synthesizingor expressing polypeptide fragments containing various Nope sequences ordeletions, the Nope binding interface can be readily identified.

[0083] The invention also provides a method of identifying non-cellularmolecules, or Nope modulatory compounds, that modulate Nope expressionor activity. A Nope modulatory compound is a molecule that specificallybinds a Nope nucleic acid molecule or Nope polypeptide and alters itsexpression or activity. A Nope modulatory compound can be a naturallyoccurring macromolecule, such as a peptide or polypeptide, nucleic acid,carbohydrate, lipid, or any combination thereof. A Nope modulatorycompound also can be a partially or completely synthetic derivative,analog or mimetic of such a macromolecule, or a small organic orinorganic molecule prepared partly or completely by combinatorialchemistry methods.

[0084] Methods for producing pluralities of compounds to use inscreening for Nope modulatory compounds, including chemical orbiological molecules such as simple or complex organic molecules,metal-containing compounds, carbohydrates, peptides, proteins,peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies,and the like, are well known in the art and are described, for example,in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem.Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998);Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev.15:481-496 (1995); and the like. Libraries containing large numbers ofnatural and synthetic compounds also can be obtained from commercialsources. Combinatorial libraries of molecules can be prepared using wellknown combinatorial chemistry methods (Gordon et al., J. Med. Chem. 37:1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994);Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson and Czarnik,eds., Combinatorial Chemistry: Synthesis and Application, John Wiley &Sons, New York (1997)).

[0085] A variety of low- and high-throughput assays known in the art aresuitable for detecting specific binding interactions between a Nopenucleic acid molecule or polypeptide and a candidate Nope modulatorycompound. Both direct and competitive assays can be performed,including, for example, fluorescence correlation spectroscopy (FCS) andscintillation proximity assays (SPA) (reviewed in Major, J. ReceptorSignal Transduction Res. 15:595-607 (1995); and in Sterrer et al., J.Receptor Signal Transduction Res. 17:511-520 (1997)). Assays fordetecting specific binding interactions can include affinity separationmethods using a Nope-specific ligand, for example, an antibody used inELISA assays, FACS analysis or affinity separation.

[0086] Assays to identify compounds that modulate Nope gene expressioncan involve first transducing cells with a Nope promoter-reporternucleic acid construct such that a change in expression of a proteinsuch as β-lactamase, luciferase, green fluorescent protein orβ-galactosidase will be detected in response to contacting the cell witha Nope modulatory compound that upregulates or downregulates expressionof Nope. Such assays and reporter systems are well known in the art andare described, for example, in Ausubel et al., supra, 1999. Other assaysto identify compounds that modulate Nope gene expression include assaysthat measure levels of Nope transcripts, such as Northern blots, RNaseprotection assays, and RT-PCR. Methods of identifying a Nope promoterand/or enhancer from Nope genomic DNA are well known in the art. Areporter gene construct can be generated using the promoter region ofNope and screened for compounds that increase or decrease Nope genepromoter activity. Such compounds can also be used to alter Nopeexpression.

[0087] Assays to identify compounds that modulate Nope polypeptideexpression can involve detecting a change in Nope polypeptide abundancein response to contacting the cell with a Nope modulatory compound.Assays for detecting changes in polypeptide expression include, forexample, immunoassays with specific Nope antibodies, such asimmunoblotting, immunofluorescence, immunohistochemistry andimmunoprecipitation assays.

[0088] Appropriate assays to determine whether a Nope modulatorycompound inhibits or promotes Nope activity can be determined by thoseskilled in the art based on the biological activity of Nope as describedbelow. For example, Nope can be screened with various compounds, asdescribed above, to identify a Nope modulatory compound that altersexpression of a Nope nucleic acid or polypeptide or that alters abiological activity of a Nope polypeptide.

[0089] The Nope polypeptides and nucleic acid molecules of the inventioncan be used in various diagnostic or therapeutic applications. Thediagnostic and therapeutic applications can be based on variousbiological activities of Nope, as described herein. For example, theexpression pattern of Nope, as disclosed herein, indicates that Nope canbe involved in neurogenesis and proliferation control. Therefore, a Nopemodulatory compound can be used to alter proliferative activity of Nope.The skilled artisan appreciates that molecular pathways involved in cellproliferation are generally well conserved among eukaryotic ogransisms.Therefore, a proliferation assay can be performed in any eukaryotic celltype in which altered proliferation can be detected including, forexample, primary mammalian cells, normal and transformed mammalian celllines, yeast, insect cells and amphibian cells. For example, a Nopenucleic acid can be transfected into a cell and a Nope polypeptideexpressed recombinantly. The transfected cell containing Nope can bescreened with various compounds, as described herein, to identify a Nopemodulatory compound that alters a proliferative response of Nope.

[0090] As disclosed herein, Nope is homologous to DCC, which wasinitially thought to be a tumor suppressor because it is absent orreduced in expression in most late-stage human colon tumors (Kolodziej,Curr. Opin. Genet. Dev. 7:87-92 (1997). Alteration of DCC expressionoccurs late in tumor progression and is likely to be reduced duringtumor progression. Inactivation of DCC also occurs in several othertumor types, including gastric, pancreatic, endometrial, breast,prostate, esophageal, bladder and squamous cell cancers (Fearon andPierceall, Cancer Surveys 24:3-17 (1995)).

[0091] Based on the homology of Nope with DCC, it is possible that Nopecan function as a tumor suppressor. If Nope can function as a tumorsuppressor, the methods of the invention can be used as diagnosticmethods to identify an individual predisposed to developing cancer, forexample, by detecting reduced expression of a Nope nucleic acid moleculeor Nope polypeptide by the methods disclosed herein. The diagnosticmethods described herein can also be used to identify individuals atincreased risk of developing a proliferative disease, such as cancer,due to hereditary mutations in a Nope gene.

[0092] Furthermore, if a Nope activity or loss thereof is associatedwith tumorigenesis, a tumor can be staged by determining changes inexpression of a Nope nucleic acid molecule or polypeptide associatedwith a cancer. As such, the diagnostic methods described herein can alsobe adapted for use as prognostic assays. Such an application takesadvantage of the observation that alterations in expression or structureof different tumor suppressor molecules can take place at characteristicstages in the progression of a proliferative disease or of a tumor. If acorrelation can be determined between Nope expression and the stage of atumor, such knowledge can be used by the clinician to select the mostappropriate treatment for the tumor and to predict the likelihood ofsuccess of that treatment. One skilled in the art can readily determinea correlation between Nope expression and the stage of a tumor bymeasuring the expression of Nope at various stages of tumor developmentusing the methods disclosed herein and determining such a correlation.

[0093] Bardet-Biedl syndrome is an autosomal recessive disordercharacterized by mental retardation, obesity, polydactyly, retinitispigmentosa and hypogonadism (Carmi et al., Human Mol. Gen. 4:9-13(1995)). Patients with this disorder also have a high incidence ofhypertension, diabetes mellitus, and renal and cardiovascular anomalies.As disclosed herein, the Nope gene is located on chromosome 15, and the3′-untranslated region of the Nope gene showed sequence homology to twohuman STS marker, WI-18508 and WI-16786, which have been mapped close toa locus on chromosome 15 that is linked to Bardet-Biedl syndrome. Nopeis expressed in the hippocampus, an area of the brain associated withcognitive functions such as learning and memory. Since Bardet-Biedlsyndrome is associated with mental retardation, it is possible thataltered Nope expression or activity, or altered expression or activityof a gene linked to Nope, can be associated with Bardet-Biedl syndrome.If an association between Nope or a Nope-linked gene and Bardet-Biedlsyndrome is determined, the Nope nucleic acid molecules of the inventioncan be used to diagnose Bardet-Biedl syndrome.

[0094] In addition, a Nope nucleic acid molecule can be used intherapeutic methods to treat an individual having an altered Nopeactivity. An altered Nope activity that is decreased relative to normalNope expression can be compensated, for example, by increasingexpression of Nope by administering a nucleic acid encoding Nope.Accordingly, a decrease or loss of an activity associated with Nope canbe compensated by administering a Nope nucleic acid in an expressionvector that allows expression of a Nope polypeptide. Alternatively, analtered Nope activity that is increased relative to normal Nopeexpression can be decreased by administering a Nope anti-sense nucleicacid.

[0095] For example, loss of a tumor suppressor activity associated withNope that has been decreased or eliminated in a tumor can beadministered to the tumor to restore tumor suppressor activity. Ifaltered Nope activity is associated with Bardet-Biedel syndrome, theNope activity can also be altered by either increasing expression ofNope by introducing a nucleic acid encoding Nope or by decreasingexpression of Nope using an anti-sense nucleic acid. A vector containinga Nope nucleic acid molecule can be introduced into an individual by invivo or ex vivo methods to restore or increase expression of a Nopepolypeptide. Vectors useful for such therapeutic methods include, forexample, retrovirus, adenovirus, lentivirus, herpesvirus, poxvirus DNAor any viral DNA that allows expression of the heterologouspolynucleotide of interest. Other vectors can also be employed, forexample, DNA vectors, pseudotype retroviral vectors, adeno-associatedvirus, gibbon ape leukemia vector, vesicular stomatitis virus (VSV),VL30 vectors, liposome mediated vectors, and the like.

[0096] Nope modulatory compounds can also be used in therapeuticmethods. For example, a Nope modulatory compound can be used to alterthe expression or activity of a Nope polypeptide that is aberrantlyexpressed or has aberrant activity. For example, excessive proliferativeactivity associated with Nope can be reduced with a Nope modulatorycompound that decreases expression of Nope or decreases Nopeproliferative activity. If an altered Nope activity is associated withBardet-Biedl syndrome, Nope modulatory compounds can be used to increaseor decrease Nope activity, as appropriate, to treat signs or symptomsassociated with Bardet-Biedl syndrome.

[0097] The present invention further provides transgenic non-humanmammals that are capable of expressing exogenous nucleic acids encodinga Nope polypeptide. An exogenous nucleic acid refers to a nucleic acidsequence which is not native to the host, or which is present in thehost in other than its native environment, for example, as part of agenetically engineered DNA construct. In addition to naturally occurringlevels of Nope, a Nope polypeptide of the invention can either beoverexpressed or underexpressed in transgenic mammals, for example, asin the well-known knock-out transgenics.

[0098] Also contemplated herein, is the use of homologous recombinationof mutant or normal versions of Nope genes with the native gene locus intransgenic animals to alter the regulation of expression or thestructure of Nope polypeptides by replacing the endogeneous gene with arecombinant or mutated Nope gene. Methods for producing a transgenicnon-human mammal including a gene knock-out non-human mammal, are wellknown to those skilled in the art (see, Capecchi et al., Science244:1288 (1989); Zimmer et al., Nature 338:150 (1989); Shastry,Experentia, 51:1028-1039 (1995); Shastry, Mol. Cell. Biochem.,181:163-179 (1998); and U.S. Pat. No. 5,616,491, issued Apr. 1, 1997,U.S. Pat. No. 5,750,826, issued May 12, 1998, and U.S. Pat. No.5,981,830, issued Nov. 9, 1999).

[0099] Also provided are transgenic non-human mammals capable ofexpressing nucleic acids encoding a Nope polypeptide so mutated as to beincapable of normal activity and which, therefore, do not express nativeNope. The present invention also provides transgenic non-human mammalshaving a genome comprising antisense nucleic acids complementary tonucleic acids encoding a Nope polypeptide, placed so as to betranscribed into antisense mRNA complementary to mRNA encoding a Nopepolypeptide, which hybridizes to the mRNA and, thereby, reduces thetranslation thereof. The nucleic acid can additionally comprise aninducible promoter and/or tissue specific regulatory elements, so thatexpression can be induced, or restricted to specific cell types.Examples of nucleic acids are DNA or cDNA having a coding sequencesubstantially the same as the coding sequences shown in SEQ ID NO:1. Anexample of a non-human transgenic mammal is a transgenic mouse. Examplesof tissue specificity-determining elements are the metallothioneinpromoter and the L7 promoter.

[0100] Animal model systems that elucidate the physiological andbehavioral roles of Nope polypeptides are also provided and are producedby creating transgenic animals in which the expression of the Nopepolypeptide is altered using a variety of techniques. Examples of suchtechniques include the insertion of normal or mutant versions of nucleicacids encoding a Nope polypeptide by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal (see, forexample, Hogan et al., Manipulating the Mouse Embryo: A LaboratoryManual (Cold Spring Harbor Laboratory, (1986)).

[0101] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso provided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cloning and Sequence Analysis of Nope

[0102] This example describes cloning and sequence analysis of the mouseNope gene.

[0103] A novel mouse gene encoding a protein of the immunoglobinsuperfamily was identified by positional cloning on chromosome 9. In thecourse of experiments designed to understand the regulation of the mousegene Punc, the DNA sequence of the genomic region starting at a BamHIsite located 7.3 kb upstream of the Punc ATG start codon and ending atthe second exon of Punc was determined. Analysis of the Punc 5′-upstreamregion revealed that a series of mouse, rat, and human ESTs were eitheridentical or similar to genomic DNA sequences of the Punc gene locus(FIG. 1A). In FIG. 1A, the location of ESTs in the genomic regionupstream of the Punc gene are depicted with black bars, and thecorresponding Genbank Accession numbers are shown: mouse (AA389134,AA051759, W33247,W83755, AI154094); rat (AA944556); and human(AI693740). The ell region indicates the cloned restriction fragmentused to generate a Nope hybridization probe. The Nope polyadenylationsignal and the ATG start codon of the Punc gene are shown.

[0104] Analysis of the region 5′ of Punc revealed the presence ofanother gene close to the Punc gene. The polyadenylation site of theputative transcript was located 3.5 kb upstream of the Punc start codon.The transcriptional direction of the new gene was identical to that ofPunc.

[0105] Using an EcoRV-Dra III restriction fragment from this region 5′of Punc to generate an antisense riboprobe, hybridization experimentswere carried out to determine whether this genomic region wastranscribed. Briefly, for northern blot hybridization, RNA was extractedfrom adult mouse tissues using TRIZOL (Life Technologies; GaithersburgMd.). Five μg RNA from each tissue was separated by gel electrophoresis,blotted onto a nylon membrane, and hybridized with a digoxigenin(DIG)-labeled (Boehringer Mannheim; Indianapolis Ind.) antisenseriboprobe transcribed from a cloned 1.8 kb DraIII/EcoRV restrictionfragment covering the 3′-untranslated area of the Nope gene (position3819 to 5682). Hybridization signals were visualized bychemiluminescence.

[0106] Northern blot analysis on RNA samples prepared from adult mousetissues revealed a single transcript of 6.5 kb detected by the probederived from the 3′-untranslated region of the Nope gene. Nopeexpression was detected in brain, cerebellum, heart, and skeletalmuscle. Nope expression was not detected in lung, liver, spleen, orkidney. These results indicated that a gene located upstream of the Puncgene is expressed and corroborated the EST data in public databases. Dueto its genomic location, the novel gene was named Nope, which stands forNeighbor of Punc e11 (e11: probe plasmid).

[0107] Sequence analysis of the genomic fragment indicated the presenceof a small reading frame without any discernible similarities to knownprotein sequences. Comparing the length of the putative 3′-untranslatedregion contained in the genomic sequence to the apparent length of theNope mRNA as seen on the Northern blot revealed that the mRNA extendedfor about 3.8 kb in the 5′ direction. Cloning cDNA sequences that spanthis region was achieved with two steps of RACE (rapid amplification ofcDNA ends) technology, using polyA⁺ RNA preparations from adult mousemuscle and from mouse embryos at 11.5 days of gestation.

[0108] Briefly, for cloning of cDNA derived from the Nope transcript,RNA was extracted from mouse embryos at 11.5 days of gestation or fromadult mouse skeletal muscle tissue using TRIZOL (Life Technologies) andsubjected to two cycles of oligo (dT) chromatography. cDNA wassynthesized from 0.5 μg polyA⁺ RNA with Superscript II or Thermoscriptreverse transcriptase (Life Technologies) and specific primers. cDNA wasamplified by RACE or ligation-mediated anchor PCR procedures. Thefollowing oligonucleotide primers were used in two RACE steps togetherwith reagents from RACE system (Life Technologies): cDNA synthesis step1,5′-AAGCAGGTGAGCCTCTCTGGCCCACT-3′ (SEQ ID NO:25)(position 3599 in cDNAsequence); amplification 1,5′-CTTGAGACAGATCCACAGCTCCAGAC-3′ (SEQ IDNO:26)(position 3526); nested amplification1,5′-ATCCGGGAAGGGCTTCCCTGTGGGAGCTTC-3′ (SEQ ID NO:27)(position 2965);cDNA synthesis step 2,5′-GCGCTGGGGACATCGTCCAGTGTATG-3′ (SEQ IDNO:28)(position 1583); amplification2,5′-GTTCCAGGTCCCGAACCTGCAGCTCTGT-3′ (SEQ ID NO:29)(position 1480);nested amplification 2,5′-CCACTCCCCTTGCCTTTTGGTAGTGAA-3′ (SEQ IDNO:30)(position 1414). Amplification products were cloned (Invitrogen;Carlsbad Calif.) and sequenced using Thermo Sequenase (AmershamPharmacia Biotech; Piscataway N.J.).

[0109] The sequence obtained covers a total of 6.1 kb, including the3′-untranslated region (UTR). Attempts at obtaining further extensionwere unsuccessful. The difference in the obtained size of 6.1 kbcompared to the apparent mRNA size of 6.5 kb was presumed to reside insequences located further upstream as well as a polyadenosine tail.Conceptual translation of the cDNA sequence revealed an open readingframe of 1244 amino acids but no start codon.

[0110] For genomic cloning, HindIII-StuI and HindIII-EcoRV restrictionfragments from a BAC clone (Genome Systems; St. Louis Mo.) covering theNope gene were identified by Southern blot hybridization withDIG-labeled (Boehringer) oligonucleotides 5′-GTGCTGACCTTCTGCCTGCTG-3′(SEQ ID NO:31)(cDNA position 34) and 5′-CTCTGTCTGCTACACTGGTCAAC-3′ (SEQID NO:32)(located in the 3′-end of intron 1), cloned and sequenced. ThecDNA sequence for the Nope mRNA is shown in FIG. 2A and is accessibleunder Genbank accession number AF176694. The sequence of genomicsequence encoding the first exon of Nope is shown in FIG. 2C.

[0111] Genomic cloning and sequencing of the relevant area from a BAC(bacterial artificial chromosome) clone demonstrated the presence of asingle ATG codon 23 bp upstream of the starting base of the longest cDNAclone. The genomic sequence displays an extremely high GC content of 82%in 400 bp upstream of a splice donor site, possibly accounting forreverse transcriptase extension problems on this sequence.

[0112] Using the ATG identified in the genomic clone as the start codonfor translation yielded a novel protein sequence of 1252 amino acids(FIG. 2B). Protein domain analysis of the Nope Sequence revealed thepresence of a signal peptide, four immunoglobulin (Ig) domains, fivefibronectin-type III (FnIII) repeats, a transmembrane domain and acytoplasmic domain of 274 amino acids (see FIG. 1B). FIG. 1C shows thedomain structure of the Nope protein in comparison to DCC, Punc, andNCAM. Shading indicates similarity among Ig domains. The domainstructure indicates that the Nope protein most closely resembles celladhesion molecules.

[0113] These results show that a novel gene, termed Nope, is a member ofthe immunoglobulin superfamily and is structurally related to celladhesion molecules, in particular Punc and DCC.

EXAMPLE II Developmental Expression of Nope mRNA

[0114] This example describes the tissue distribution and developmentalexpression of Nope mRNA as detected by in situ hybridization.

[0115] To determine the tissue distribution and developmental expressionof Nope, in situ hybridization was performed on developing mouseembryos. Briefly, in situ hybridization was performed using DIG-labeledantisense riboprobes on 25 μm cryosections prepared from frozen tissuesessentially as described previously (Long and Salbaum, Mol. Biol. Evol.15:284-292 (1998)). A probe for myosin heavy chain was derived form anEST (Genbank accession number AF200922).

[0116] In situ hybridization analyses with a Nope antisense riboprobedemonstrated that Nope is expressed in the developing mouse embryo. Thefirst weak signals of Nope expression can be detected in the notochordat 9.5 days of gestation (E9.5). In a cross section through the cervicalregion of a mouse embryo at E10.5, the main expression domain of Nope isin developing muscle tissues, starting in the dermomyotome of somites atE10.5 and is evident in the developing muscles of the forelimb and thebody wall. A section through the cervical region of a mouse embryo atE15.5 revealed expression in skeletal muscles and in the nervous system.Nope expression in notochord is still visible at E10.5. Nope expressionincreases with the growth of muscles. In a cervical section at E15.5,hybridization signals for Nope can be found in all skeletal muscles,similar to a hybridization signal for the embryonic form of myosin heavychain. In addition to muscle-specific expression, a Nope signal can bedetected at E15.5 in the developing nervous system. A coronal sectionthrough the head of an embryo at E15.5 revealed Nope expression in theventricular zone of the forebrain. Nope expression was observed in thedeveloping muscles at E15.5 with a perinuclear localization of thetranscript, which resulted in a “doughnut”-shaped appearance of thestaining on cross sections. Nope expression is concentrated in theventricular zone in the brain, a region containing proliferatingneuroblasts as well as developing glial cells. In a section of an adultbrain, Nope expression can be found in the hippocampus, the piriformcortex, thalamic nuclei, and foliae of the cerebellum. This expressionpattern suggests that Nope initially contributes to the cell surfaceproperties of developing muscle cells and functions in cells of thenervous system that arise late in gestation.

[0117] These results show that Nope expression increases duringdevelopment and that Nope is expressed in the notochord, developingmuscle tissue and in specific regions of the brain, including theventricular zone, hippocampus, piriform cortex, thalamic nuclei andcerebellum.

EXAMPLE III Evolutionary Relationship Between Nope and ImmunoglobulinSuperfamily Members

[0118] This example describes the evolutionary relationship between Nopeand various members of the immunoglobulin superfamily.

[0119] To characterize the relationship between Nope and other knownsequences, protein database searches were performed to analyze sequencerelationships between Nope and other proteins. Sequence analysis andassembly was performed using MacMolly software (SoftGene; Berlin,Germany). On-line database searches were performed using BLAST (Altschulet al., Nucleic Acids Res. 25:3389-3402 (1997)), and domain analysis ofthe Nope protein sequence was carried out using SMART (Schultz et al.,Proc. Natl. Acad. Sci. USA 95:5857-5864 (1998)). Signal peptides werepredicted using the program SignalP (Nielsen et al., Protein Engineering10:1-6 (1997)).

[0120] Analysis of the evolutionary relationships between theimmunoglobulin domains of Nope and related proteins of theimmunoglobulin superfamily was done with the program PAUP (SinauerAssociates; Sunderland Mass.). Alignments were constructed either fromregions spanning a total of four Ig domains or from sequencesrepresenting individual single Ig domains. In the case of NCAM and L1,the four Ig domains closest to the FnIII repeats were selected. Asoutgroup in this analysis, a heavy chain variable domain sequencederived from an antibody against hepatitis B Virus X protein was chosen(Genbank Accession No. AAC82376). Dot plot representations of sequencecomparisons between Nope and Punc are based on PAM matrix and wereconstructed (match length 20, up to 8 mismatches) using MacMollyComplign software (SoftGene).

[0121] Nope is most similar to Punc, with 45% sequence identity in theregion ranging from the beginning of the second Ig domain throughout thefirst FnIII repeat. Other protein sequences that were similar to Nopeare Neogenin (Meyerhardt et al., Oncogene 14:1129-1136 (1997);Vielmetter et al., Genomics 41:414-421 (1997)) and DCC (Fearon et al.,Science 247:49-46 (1990)). Nope therefore belongs to a subfamily of theimmunoglobulin superfamily of proteins that is characterized by thepresence of four Ig domains and consists of vertebrate DCC, Neogenin,and Punc, Drosophila frazzled, and UNC-40 of C. elegans. DCC, frazzledand UNC-40 can all function as axonal guidance receptors (Chan et al.,Cell 87:187-195 (1996); Keino-Masu et al., Cell 87:175-185 (1996);Kolodziej et al., Cell 87:197-204 (1996)). Therefore, Nope likely has asimilar function as an axonal guidance receptor.

[0122] Analysis of sequence relationships of Nope, Punc, and otherguidance receptors or neuronal cell adhesion molecules demonstrated thatboth Nope and Punc are more similar to the DCC subgroup of the Igsuperfamily than they are to classical neuronal cell adhesion moleculessuch as NCAM (Cunningham et al., Science 236:799-806 (1987)) or L1(Kobayasi et al., Biochem. Biophys. Acta 1090:238-240 (1991))(FIG. 3A).Within this branch, Nope and Punc group closely together, as do DCC andNeogenin, whereas sequences from invertebrate species appear to be moredistantly related.

[0123] Examination of individual Ig domains from vertebrate proteinsequences revealed that, within the DCC subgroup, cognate domains fromdifferent proteins are more similar than adjacent domains within thesame molecule (FIG. 3B), as demonstrated by the grouping together of thefirst, third, and fourth Ig domains of DCC, Neogenin, Nope and Punc.Within these branches, relationships are conserved, since DCC andNeogenin sequences are closely related, as are sequences from Nope andPunc. In contrast, domains from the neural cell adhesion molecule NCAMare more similar to each other than they are to domains from proteins ofthe DCC subgroup.

[0124] While the close relationship between Nope and Punc could beclearly demonstrated for large parts of the extracellular domain, theclose relationship does not extend to the whole protein sequence (FIG.3C). FIG. 3C shows the sequence relationship between Nope and Punc asanalyzed by dot plot analysis based on a PAM similarity matrix. Only theregion containing the Ig domains and the first adjacent FnIII repeat arehighly similar between the two proteins. The respective cytoplasmicdomains of Nope and Punc did not exhibit sequence similarity to eachother, as indicated by the lack of diagonal lines in this region, anddatabase searches did not yield any other protein motif or domainmatching Nope or Punc. Therefore, Nope is related to guidance receptorsof the DCC family and can participate in similar extracellularinteractions while containing a novel, diverse intracellular signalingdomain.

[0125] The human gene for Neogenin has previously been mapped tochromosome 15 in the band 15q22.3-23 (Meyerhardt et al., supra, 1997;Vielmetter et al., supra, 1997), and human PUNC has been placed on thesame band of chromosome 15 (Salbaum, Mamm. Genome 10:107-111 (1999)). Todetermine whether a similar colocalization exists in the mouse and toobtain high resolution positional information for the Nope gene,Neogenin, Nope, Punc, and two BAC end sequences were mapped usingradiation hybrid mapping on the mouse T31 panel (McCarthy et al., GenomeRes. 7:1153-1161 (1997)).

[0126] Briefly, for radiation hybrid mapping, DNA from the T31 radiationhybrid panel (McCarthy et al., supra, 1997) was obtained from ResearchGenetics and used for mapping experiments. The following oligonucleotideprimer pairs were used: Punc (from genomic DNA sequence),5′-TGGACGCCAAGGAGTTGG-3′ (SEQ ID NO:33), and 5∝-CAAATCCCACAGAACAGGA-3′(SEQ ID NO:34), amplicon size 1236 bp; Nope (upstream primer derivedfrom genomic DNA, downstream primer from cDNA sequence at position2975), 5′-ACGGGCATCATCGTGGG-3′ (SEQ ID NO:35) and5′-GAGGAGGACAATCCGGGAAGGGCTT-3′ (SEQ ID NO:36), amplicon size 592 bp;Neogenin (from cDNA genbank Acc. No. Y09535, position 5107 to 5367),5′-TCAAGCAGTTGACACTTGACTGTG-3′ (SEQ ID NO:37) and5′-TAATCTCACAGTGATGAGAGGAGA-3′ (SEQ ID NO:38), amplicon size 260 bp;296S, 5′-CTGTGTCTCAATCTTGAACAAACACA-3′ (SEQ ID NO:39) and5′-GGAAGAGAGACAGTAAACATTTCGT-3′ (SEQ ID NO:40), amplicon size 266 bp;331T, 5′-CTCCCTTCCTTCCTGATCGTTTTC-3′ (SEQ ID NO:41) and5′-CGGCTCTCAAGCACTGCAGATTTTG-3′ (SEQ ID NO:42), amplicon size 111 bp.The marker 296S was derived from the end sequence of a BAC clonecontaining the Nope gene and is located 5′-upstream of Nope; the marker331T was derived from an overlapping BAC clone containing part of theNope gene and all of the Punc gene (Salbaum, supra, 1999) and is located3′-downstream of the Punc gene. Hot start PCR conditions were 94° C. for3 min, followed by 35 cycles of 93° C. for 30 seconds, 58° C. for 30seconds, and 72° C. for 75 seconds, and finished at 72° C. for 5minutes. Assays for each primer pair were performed in triplicate,analyzed by agarose gel electrophoresis, and summarized as data vectors.Results were analyzed using on-line mapping software provided by theWhitehead Institute Center for Genome Research(http://www.genome.wi.mit.edu/mouse_rh/index.html), which allowedplacement of all markers relative to framework markers on mousechromosome 9.

[0127] PCR assays with primer pairs specific for each gene or markerwere carried out using DNA from the T31 panel. The results of the PCRassays were summarized as data vectors, which are shown in Table 1. “1”indicates the presence, “O” indicates the absence, and “2” indicates theweak presence of a PCR product. The T31 panel shown in Table 1represents 100 cell lines in which fragments of irradiated mouse genomicDNA were introduced by fusion with hamster cells. The total panelrepresents the whole mouse genome. Based on the pattern of hybridizationand comparison to known markers, Nope was found to map to chromosome 9.TABLE 1 Gene/Marker T31 data vector: Punc 00000 01210 20002 00000 0002001010 20000 00001 10000 00102 10000 10101 02010 00010 10000 10001 1000000000 01001 00001 Nope 00000 01210 00002 00000 00020 01010 10010 0000110000 00101 10000 10101 00010 00210 10000 10001 10002 00200 01001 00001Ngn 00000 00010 00110 00000 00020 01010 10000 00001 10000 00102 0001020101 00010 00200 00010 10101 11000 00000 02101 00000 331T 00000 0111000002 00000 00020 01020 20020 00001 10000 00101 10000 00001 00210 0200000000 00000 00000 00000 00000 00000 296S 00000 01210 00002 01000 0002001012 10020 00021 10000 00102 10000 10101 00020 00210 00000 20002 2000000000 02002 00001

[0128] Neogenin maps very close to both Nope and Punc on chromosome 9(FIG. 4). Localization of the genes for Nope, Punc and Neogenin on mousechromosome 9 are shown in FIG. 4. Structures of the encoded proteins areindicated next to the chromosome sketch. Placement of Neogenin, Nope,Punc, and BAC end markers relative to framework markers D9Mit48 andD9Mit143 on chromosome 9 are indicated. Distances are given in cR. Thearrangement of BAC clones and the origin of PCR products used formapping is shown on the right.

[0129] Neogenin showed linkage to the framework marker D9Mit48 at 31.7cM, whereas Nope, Punc, and both BAC end markers were found to be linkedto the framework marker D9Mit143, which is also placed at 31.7 cM.Neogenin therefore maps to a region of mouse chromosome 9 that issyntenic to the region on human chromosome 15 where Neogenin had beenplace on the cytogenetic map. Radiation hybrid mapping of Nope and Puncconfirmed previous cytogenetic mapping experiments and established alink between the radiation hybrid and cytogenetic map of mousechromosome 9.

[0130] Based on the similarity to DCC, Punc and Nope are members of asubgroup of the DCC Ig superfamily, termed herein DEAL, for DCC et al.The high sequence similarity between Punc and Nope, identical directionof transcription, and close physical proximity initially suggested thatthese two genes likely arose through a gene duplication event. Themapping data place the gene for Neogenin, a core member of the Dealsubgroup, in the immediate vicinity of the genes for Nope and Punc.These genes are therefore clustered on chromosome 9. In light of thefinding that C. elegans, and, as of this time Drosophila, have only oneDEAL gene in their respective genomes, it is likely that the genecluster on chromosome 9 in the mouse is the result of a recent linearexpansion of the DEAL family that occurred only in the vertebratelineage. Given the closer relationship of DCC and Neogenin to theinvertebrate counterparts, Neogenin represents a more ancestral gene inthe cluster, and Nope and Punc are more derived. If so, the divergenceof Punc and Nope included sequence variation and domain loss, possiblythrough intragenic recombination events. Further mapping in this regionof chromosome 9 will reveal whether there are additional members of thisgene cluster.

[0131] In contrast to the structural similarities between these DEALgenes on chromosome 9, expression is strikingly different. The Punc geneis expressed shortly after gastrulation in the neuroectoderm, and itsexpression domain includes early proliferating cells in the developingnervous system and the lateral plate mesoderm. After mid-gestation, Puncundergoes a down regulation that appears unusual for Ig CAMs. Incomparison to Punc, Nope expression increases as development progressesand persists in the adult animal. Such an overall pattern is morecommonly observed with Ig superfamily genes and typically attributed tothe fact that cell surface properties of differentiated cells are moreelaborate compared to proliferating precursor cells. Nope and Puncdiffer not only in their temporal but also in their cellular specificityof expression, suggesting that divergence of genes in this region is notrestricted to coding regions but includes regulatory elements. Thislatter point is of particular interest given the close proximity ofthese two genes. Identification of regulatory sequences of Nope and Puncis useful for understanding the specificity of expression of these genesin developmental gene regulation.

[0132] The 3′-untranslated region of the Nope gene showed sequencehomology to two human STS markers, WI-18508 and WI-16786 (Salbaum,supra, 1999). Both markers have been mapped close to a locus on achromosome 15 which is linked to Bardet-Biedl syndrome 4 (Carmi et al.,Hum. Mol. Genet. 4:9-13 (1995)). Considering that mental retardation ispart of Bardet-Biedl syndrome, it was intriguing to detect Nope geneexpression in the adult hippocampus, an area of the brain associatedwith cognitive functions such as learning and memory. Therefore, it ispossible that the Nope gene plays a role in related human disorders.

[0133] Throughout this application various publications have beenreferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

[0134] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

1 45 1 6176 DNA Mus musculus CDS (1)...(3756) 1 atg gcg cgg gcg gac acgggc cgc ggg ctc ctg gtg ctg acc ttc tgc 48 Met Ala Arg Ala Asp Thr GlyArg Gly Leu Leu Val Leu Thr Phe Cys 1 5 10 15 ctg ctg tcc gcg cgc ggggag ctg cca ttg ccc cag gag aca act gtc 96 Leu Leu Ser Ala Arg Gly GluLeu Pro Leu Pro Gln Glu Thr Thr Val 20 25 30 aag ctg agc tgt gat gag ggaccc ctg caa gtg atc ctg ggc cct gag 144 Lys Leu Ser Cys Asp Glu Gly ProLeu Gln Val Ile Leu Gly Pro Glu 35 40 45 cag gct gtg gtg ctg gac tgc actttg ggg gct aca gct gct ggg cct 192 Gln Ala Val Val Leu Asp Cys Thr LeuGly Ala Thr Ala Ala Gly Pro 50 55 60 ccg acc agg gtg aca tgg agc aag gatgga gac act gta cta gag cat 240 Pro Thr Arg Val Thr Trp Ser Lys Asp GlyAsp Thr Val Leu Glu His 65 70 75 80 gag aac ctg cac ctg cta ccc aat ggctcc ctg tgg ctg tcc tca ccc 288 Glu Asn Leu His Leu Leu Pro Asn Gly SerLeu Trp Leu Ser Ser Pro 85 90 95 cta gag caa gaa gac agc gat gat gag gaagct ctt agg atc tgg aag 336 Leu Glu Gln Glu Asp Ser Asp Asp Glu Glu AlaLeu Arg Ile Trp Lys 100 105 110 gtc act gag ggc agc tat tcc tgt ctg gcccac agc ccg cta gga gtg 384 Val Thr Glu Gly Ser Tyr Ser Cys Leu Ala HisSer Pro Leu Gly Val 115 120 125 gtg gcc agc cag gtt gct gtg gtc aag cttgcc aca ctc gaa gac ttc 432 Val Ala Ser Gln Val Ala Val Val Lys Leu AlaThr Leu Glu Asp Phe 130 135 140 tct ctg cac ccc gag tcc cag att gtg gaggag aac ggg aca gca cgc 480 Ser Leu His Pro Glu Ser Gln Ile Val Glu GluAsn Gly Thr Ala Arg 145 150 155 160 ttt gaa tgc cac acc aag ggc ctt ccagcc ccc atc att act tgg gaa 528 Phe Glu Cys His Thr Lys Gly Leu Pro AlaPro Ile Ile Thr Trp Glu 165 170 175 aag gac cag gtg acc gtg cct gag gagccc cgg ctc atc act ctt ccc 576 Lys Asp Gln Val Thr Val Pro Glu Glu ProArg Leu Ile Thr Leu Pro 180 185 190 aag tgg ctc ctc cag atc cta gat gtccag gac agt gat gca ggc tcc 624 Lys Trp Leu Leu Gln Ile Leu Asp Val GlnAsp Ser Asp Ala Gly Ser 195 200 205 tac cgc tgc gtg gcc acc aat tca gcccgc caa cga ttc agc cag gag 672 Tyr Arg Cys Val Ala Thr Asn Ser Ala ArgGln Arg Phe Ser Gln Glu 210 215 220 gcc tcg ctc act gtg gcc ctc aga gggtct ttg gag gct acc agg ggg 720 Ala Ser Leu Thr Val Ala Leu Arg Gly SerLeu Glu Ala Thr Arg Gly 225 230 235 240 cag gat gtg gtc att gtg gca gcccca gag aac acc acg gta gtg tct 768 Gln Asp Val Val Ile Val Ala Ala ProGlu Asn Thr Thr Val Val Ser 245 250 255 gga cag aat gta gtg atg gag tgcgtg gcc tct gct gac ccc acc cct 816 Gly Gln Asn Val Val Met Glu Cys ValAla Ser Ala Asp Pro Thr Pro 260 265 270 ttt gtg tcc tgg gtc cga cag gatgga aag cct atc tcc acg gat gtc 864 Phe Val Ser Trp Val Arg Gln Asp GlyLys Pro Ile Ser Thr Asp Val 275 280 285 atc gtt ctg ggc cgg acc aat ctactc atc gcc agc gcg cag cct cgg 912 Ile Val Leu Gly Arg Thr Asn Leu LeuIle Ala Ser Ala Gln Pro Arg 290 295 300 cac tct gga gtc tat gtc tgc cgagcc aac aag ccc ctc acg cgt gac 960 His Ser Gly Val Tyr Val Cys Arg AlaAsn Lys Pro Leu Thr Arg Asp 305 310 315 320 ttc gcc act gcg gct gct gagctc cga gtg ctt gct gcc cca gcc atc 1008 Phe Ala Thr Ala Ala Ala Glu LeuArg Val Leu Ala Ala Pro Ala Ile 325 330 335 tcg cag gca ccc gag gcg ctctcg cgg acg cgg gcc agc acc gcg cgc 1056 Ser Gln Ala Pro Glu Ala Leu SerArg Thr Arg Ala Ser Thr Ala Arg 340 345 350 ttc gtg tgc cgg gcg tcc ggggag cca cgg ccc gcg ctg cac tgg ctg 1104 Phe Val Cys Arg Ala Ser Gly GluPro Arg Pro Ala Leu His Trp Leu 355 360 365 cac gac ggg atc ccg ttg cgaccc aat ggg cgc gtc aag gtg cag ggc 1152 His Asp Gly Ile Pro Leu Arg ProAsn Gly Arg Val Lys Val Gln Gly 370 375 380 ggt ggc ggc agc ttg gtc atcact cag atc ggc ctg cag gac gct ggc 1200 Gly Gly Gly Ser Leu Val Ile ThrGln Ile Gly Leu Gln Asp Ala Gly 385 390 395 400 tac tac cag tgc gta gcagaa aac agc gcg gga act gcc tgt gcc gct 1248 Tyr Tyr Gln Cys Val Ala GluAsn Ser Ala Gly Thr Ala Cys Ala Ala 405 410 415 gcg ccc ctg gcg gta gtggtg cgc gag ggg ctg ccc agc gcc ccg act 1296 Ala Pro Leu Ala Val Val ValArg Glu Gly Leu Pro Ser Ala Pro Thr 420 425 430 cgg gtc aca gcc acg ccgctg agc agc tcc tct gtg ctg gtg gcc tgg 1344 Arg Val Thr Ala Thr Pro LeuSer Ser Ser Ser Val Leu Val Ala Trp 435 440 445 gag cgg cct gag ttg cacagc gag caa atc att ggc ttc tct ctt cac 1392 Glu Arg Pro Glu Leu His SerGlu Gln Ile Ile Gly Phe Ser Leu His 450 455 460 tac caa aag gca agg ggagtg gac aat gtg gag tac cag ttt gca gta 1440 Tyr Gln Lys Ala Arg Gly ValAsp Asn Val Glu Tyr Gln Phe Ala Val 465 470 475 480 aac aat gac acc acagag ctg cag gtt cgg gac ctg gaa ccc aac acg 1488 Asn Asn Asp Thr Thr GluLeu Gln Val Arg Asp Leu Glu Pro Asn Thr 485 490 495 gat tat gag ttc tacgtg gtg gcc tac tcc cag ctg ggg gcc agc cga 1536 Asp Tyr Glu Phe Tyr ValVal Ala Tyr Ser Gln Leu Gly Ala Ser Arg 500 505 510 acc tcc agc cca gccctg gtg cat aca ctg gac gat gtc ccc agc gca 1584 Thr Ser Ser Pro Ala LeuVal His Thr Leu Asp Asp Val Pro Ser Ala 515 520 525 gca ccc cag ctt accttg tcc agc ccc aac ccc tcg gac atc agg gtg 1632 Ala Pro Gln Leu Thr LeuSer Ser Pro Asn Pro Ser Asp Ile Arg Val 530 535 540 gca tgg ctg ccc ctgccc tcc agc ctg agc aat gga cag gtg ctg aag 1680 Ala Trp Leu Pro Leu ProSer Ser Leu Ser Asn Gly Gln Val Leu Lys 545 550 555 560 tac aag ata gagtac ggt ttg ggg aag gaa gat cag gtt ttc tcc acc 1728 Tyr Lys Ile Glu TyrGly Leu Gly Lys Glu Asp Gln Val Phe Ser Thr 565 570 575 gag gtg cct ggaaat gag aca caa ctt acg tta aac tca ctt cag cca 1776 Glu Val Pro Gly AsnGlu Thr Gln Leu Thr Leu Asn Ser Leu Gln Pro 580 585 590 aac aaa gtg taccga gtc cgg att tca gct ggc act ggc gct ggc tat 1824 Asn Lys Val Tyr ArgVal Arg Ile Ser Ala Gly Thr Gly Ala Gly Tyr 595 600 605 gga gtc cct tctcag tgg atg cag cac agg aca cct ggt gtg cac aac 1872 Gly Val Pro Ser GlnTrp Met Gln His Arg Thr Pro Gly Val His Asn 610 615 620 cag agc cat gttccc ttt gcc cct gca gaa ttg aag gtg agg gca aag 1920 Gln Ser His Val ProPhe Ala Pro Ala Glu Leu Lys Val Arg Ala Lys 625 630 635 640 atg gag tccctg gtg gtg tca tgg cag ccg ccc cct cac ccc acc cag 1968 Met Glu Ser LeuVal Val Ser Trp Gln Pro Pro Pro His Pro Thr Gln 645 650 655 atc tct ggatac aaa ctc tac tgg gga gag gtg gga aca gag gag gag 2016 Ile Ser Gly TyrLys Leu Tyr Trp Gly Glu Val Gly Thr Glu Glu Glu 660 665 670 gca gat ggtgac cgc ccc cca ggg ggt cgt gga gat caa gct tgg gac 2064 Ala Asp Gly AspArg Pro Pro Gly Gly Arg Gly Asp Gln Ala Trp Asp 675 680 685 gtc ggg cccgtg cgg ctg aag aag aaa gtg aag cag tat gaa ctg acc 2112 Val Gly Pro ValArg Leu Lys Lys Lys Val Lys Gln Tyr Glu Leu Thr 690 695 700 cag tta gtccct ggc agg ccg tac gag gtg aag ctc gta gct ttc aac 2160 Gln Leu Val ProGly Arg Pro Tyr Glu Val Lys Leu Val Ala Phe Asn 705 710 715 720 aaa cacgag gac ggc tac gct gct gtg tgg aag ggc aag acg gag aag 2208 Lys His GluAsp Gly Tyr Ala Ala Val Trp Lys Gly Lys Thr Glu Lys 725 730 735 gcg cccacg cca gac ctg cct atc cag agg ggg cca ccg ctg cct cct 2256 Ala Pro ThrPro Asp Leu Pro Ile Gln Arg Gly Pro Pro Leu Pro Pro 740 745 750 gcc catgtc cac gca gag tca aac agc tcc act tcc att tgg ctt cgg 2304 Ala His ValHis Ala Glu Ser Asn Ser Ser Thr Ser Ile Trp Leu Arg 755 760 765 tgg aagaag cca gac ttt acc act gtc aag att gtc aac tac act gta 2352 Trp Lys LysPro Asp Phe Thr Thr Val Lys Ile Val Asn Tyr Thr Val 770 775 780 cgc ttcggc ccc tgg ggg ctc agg aat gct tcc ctg gtc acc tac tat 2400 Arg Phe GlyPro Trp Gly Leu Arg Asn Ala Ser Leu Val Thr Tyr Tyr 785 790 795 800 accagc tct gga gaa gac att ctc att ggc ggc ctg aaa cca ttt acc 2448 Thr SerSer Gly Glu Asp Ile Leu Ile Gly Gly Leu Lys Pro Phe Thr 805 810 815 aagtac gag ttt gcg gta cag tcc cac gga gtg gat atg gat ggg ccc 2496 Lys TyrGlu Phe Ala Val Gln Ser His Gly Val Asp Met Asp Gly Pro 820 825 830 tttggc tcc gtc gta gaa cgc tcc acc ctg cct gac cgg cct tca aca 2544 Phe GlySer Val Val Glu Arg Ser Thr Leu Pro Asp Arg Pro Ser Thr 835 840 845 cctcct tct gac ctg cgc ctg agc ccc ctg aca cca tcc acc gtt cgg 2592 Pro ProSer Asp Leu Arg Leu Ser Pro Leu Thr Pro Ser Thr Val Arg 850 855 860 ttacac tgg tgt ccc ccc acg gag ccc aat ggt gag att gtg gag tat 2640 Leu HisTrp Cys Pro Pro Thr Glu Pro Asn Gly Glu Ile Val Glu Tyr 865 870 875 880cta att ctc tac agc aac aac cac acc cag ccc gaa cac cag tgg aca 2688 LeuIle Leu Tyr Ser Asn Asn His Thr Gln Pro Glu His Gln Trp Thr 885 890 895ctg ctc acc aca gag gga aac atc ttc agt gca gag gtc cat ggc cta 2736 LeuLeu Thr Thr Glu Gly Asn Ile Phe Ser Ala Glu Val His Gly Leu 900 905 910gag agt gac act cgg tat ttc ttc aag atg gga gcc cgc aca gag gtg 2784 GluSer Asp Thr Arg Tyr Phe Phe Lys Met Gly Ala Arg Thr Glu Val 915 920 925ggg cct ggg ccc ttt tcc cgc ttg cag gat gtg att act ctg caa gag 2832 GlyPro Gly Pro Phe Ser Arg Leu Gln Asp Val Ile Thr Leu Gln Glu 930 935 940aca ttc tca gac tcc ttg gat gtg cac gcc gtc acg ggc atc atc gtg 2880 ThrPhe Ser Asp Ser Leu Asp Val His Ala Val Thr Gly Ile Ile Val 945 950 955960 ggt gtc tgc ctg ggc ctt ctc tgc ctc ctg gcc tgc atg tgt gct ggc 2928Gly Val Cys Leu Gly Leu Leu Cys Leu Leu Ala Cys Met Cys Ala Gly 965 970975 cta cga caa agc tcc cac agg gaa gcc ctt ccc gga ttg tcc tcc tca 2976Leu Arg Gln Ser Ser His Arg Glu Ala Leu Pro Gly Leu Ser Ser Ser 980 985990 ggc acc cca gga aac cca gcg ctc tac aca aga gct cgg ctt ggg cct 3024Gly Thr Pro Gly Asn Pro Ala Leu Tyr Thr Arg Ala Arg Leu Gly Pro 995 10001005 ccc agt gtc cct gct gcc cat gag ttg gag tcc ctc gtg cat cct cgt3072 Pro Ser Val Pro Ala Ala His Glu Leu Glu Ser Leu Val His Pro Arg1010 1015 1020 ccc cag gat tgg tcc cca cca ccc tca gat gtg gaa gac aaggct gaa 3120 Pro Gln Asp Trp Ser Pro Pro Pro Ser Asp Val Glu Asp Lys AlaGlu 1025 1030 1035 1040 gta cac agc ctt atg ggt ggc agt gtt tca gat tgccgg ggc cac tcc 3168 Val His Ser Leu Met Gly Gly Ser Val Ser Asp Cys ArgGly His Ser 1045 1050 1055 aag aga aag atc tcc tgg gct cag gca ggg ggacca aac tgg gca ggc 3216 Lys Arg Lys Ile Ser Trp Ala Gln Ala Gly Gly ProAsn Trp Ala Gly 1060 1065 1070 tcc tgg gca ggc tgt gag ctg ccc cag ggtagt ggt cca agg ccg gct 3264 Ser Trp Ala Gly Cys Glu Leu Pro Gln Gly SerGly Pro Arg Pro Ala 1075 1080 1085 ctg acc cgt gct ctg ctg cct cca gcggga acc ggg cag aca ctg ctg 3312 Leu Thr Arg Ala Leu Leu Pro Pro Ala GlyThr Gly Gln Thr Leu Leu 1090 1095 1100 ctg caa gcc ctg gtg tat gac ggcata aag agc aac ggg aga aag aag 3360 Leu Gln Ala Leu Val Tyr Asp Gly IleLys Ser Asn Gly Arg Lys Lys 1105 1110 1115 1120 ccg tcc cca gcc tgc aggaat cag gtg gaa gct gag gtc att gtc cac 3408 Pro Ser Pro Ala Cys Arg AsnGln Val Glu Ala Glu Val Ile Val His 1125 1130 1135 tcc gac ttc ggt gcatcc aaa gga tgt cct gac ctc cac ctc caa gac 3456 Ser Asp Phe Gly Ala SerLys Gly Cys Pro Asp Leu His Leu Gln Asp 1140 1145 1150 ctg gag cca gaggaa cca ctg act gca gag act ctg cct tcc acg tct 3504 Leu Glu Pro Glu GluPro Leu Thr Ala Glu Thr Leu Pro Ser Thr Ser 1155 1160 1165 gga gct gtggat ctg tct caa gga gca gac tgg ctg ggc agg gag ctg 3552 Gly Ala Val AspLeu Ser Gln Gly Ala Asp Trp Leu Gly Arg Glu Leu 1170 1175 1180 gga gggtgc caa cca aca acc agt ggg cca gag agg ctc acc tgc ttg 3600 Gly Gly CysGln Pro Thr Thr Ser Gly Pro Glu Arg Leu Thr Cys Leu 1185 1190 1195 1200cca gaa gca gcc agt gcc tcc tgc tcc tgc tca gac ctc cag ccc agc 3648 ProGlu Ala Ala Ser Ala Ser Cys Ser Cys Ser Asp Leu Gln Pro Ser 1205 12101215 act gct ata gag gag gcc cct ggg aaa agc tgc cag ccc aaa gcc ctg3696 Thr Ala Ile Glu Glu Ala Pro Gly Lys Ser Cys Gln Pro Lys Ala Leu1220 1225 1230 tgt cct cta aca gtc agc cca agc ctt ccc agg gcc cct gtctcc tct 3744 Cys Pro Leu Thr Val Ser Pro Ser Leu Pro Arg Ala Pro Val SerSer 1235 1240 1245 gct cag gtc ccc tgagcagaag gcagatatgg ctcaggaacatgccatgcat 3796 Ala Gln Val Pro 1250 ggctacacat gtgtgtacta gagatatccataagtccttg gagcctctta gggtcctttg 3856 gctggggttg gggagaactt tactctccctcatattctgc atcacataca ggagggactt 3916 gagacacagc tctgtgtaat ggacacgtgtgaagtcgtgt gtgtgtgtgt gtgtgtgtgt 3976 gctggttgag ctaggaaacc tctccctatgtagcactcac tgtggcctag ttgaccctcc 4036 gtggcaggat ggtgtaacag tgatcagtgccagctctttg agcttttagc cttgtcacct 4096 agccttttat tacactctga gagtgtctccagtgctgtgt ctacaaagac agcgcccagc 4156 cctcttctgt cagctgtgct gagcagagtgccagtcaact ccacgggcct atgacaccgc 4216 agcctaccac agcatggctg tcatccccctggcctcctaa ggtccagatg tctgggtgaa 4276 cccagctcag ctcccctctc ctttgagcatctctgtacct aattttgtaa tctgggaagt 4336 gcctggtttg ggaaatcttc tttcgcaccctgtccctctc tgccccttcc ttcatttgtt 4396 ctggtgatct gtctcatgtc atcttgctcgattatcctgg ggcccttctc tttcccatga 4456 tgcccctgat ttcctcactg ctgttttcatttctgtctgc catgcttgtc tttatgtcgt 4516 gtgtttctcg tccctgagtt caacctatgcaccctttcct aacaacatga ctacctcatg 4576 tctgcttcag accatagtgt gacccctgggtccccacagc tcccctgcca accgccttcc 4636 tgggcagatg agcccactcc aagtagatctggaaaagacc cttgtggctt gtctggctgc 4696 cctccccttg gtgttgagat gagaaggttttctatggaag agatgagtcc aggctgcaca 4756 ggggaacccc caagaagggg tagggagtgaaaccaagagg ctgaaaaaaa atggctgcca 4816 cccatctgca cagagagatg ggtgtgtgcttttgacgtgc agtcctggct gaaactgaag 4876 gggtgaggag aggggagcta ctggggctgccatggctcag ttccctgacc ctggagccct 4936 gaacctggct tcagagtagc aaagagtttcctccaagatg ctgtaaggga agtctttgca 4996 taggaaaagg gcggctggct cattttattttatctttctt tacactgaat cccaaaatca 5056 tcttaccaca aagggccaag cctgactggtatttcctgag tcacaagagc catgccatct 5116 ctctggtttc tcacctcagt catgtcccagaattgtcaga tccagtggca tctgtgctct 5176 tgctgcacat ctttctattt caactggctggcacatcaag tgttaactct ggcttctggg 5236 ccaagttaga aataaccagt ctattttccctttattttat tttattttat tttattttat 5296 gtctttcagt ggagttgtag cttctgaaagcgtctgtgtt tattagcctt gtgtgtcact 5356 catgtttgac cccacccaca tttccttctcctcccctctt cagccagcct atgataacac 5416 taaagattat taatgctggc ttcgtatctcattaaagaca ggattgtcac ttgaactact 5476 tctatagcat tcaaagtggc cacggccaacaccaccgtat gtttcttcat tgctctgaag 5536 gtcaagagcc tcattttgtt ttcctggttagattcttttc ctccttgcct tgaatgaaat 5596 aaccgtttta acagtaggct cttagcatcacaccacatag tcattcctca tgttcttgtt 5656 taacaagcac ttgaggttct gggtttaaattaaatagctg caaatgagac aatttataac 5716 ccattaggct gggtggaaaa ttgttctcaaaagcaaataa gtaataaatc tggtatctgc 5776 ctataactca cagttgataa gaaagtagccagaactcact agcattatat atgattgggg 5836 ttctgagtaa ctggggagtg ttagctttgtgactttgtag caccaggtct tattaggaaa 5896 gtctgttggc cttttacagg gcattagtccctttgtcgtt tgccatggat gccttaagtt 5956 ctttggagtc tcatttaaga attccttttctcgaagcatg acaagtgtat cgcaatactt 6016 acatgctcac tcgtttacct ggcttagtttgtgctgggtt atttaattgc actttccagc 6076 atcatgcttc ctccttacaa atatgatattctttattgtt acactaaggt gttgatcatg 6136 tatctgtccc tgtaaagaat taataaactattttccagac 6176 2 1252 PRT Mus musculus 2 Met Ala Arg Ala Asp Thr GlyArg Gly Leu Leu Val Leu Thr Phe Cys 1 5 10 15 Leu Leu Ser Ala Arg GlyGlu Leu Pro Leu Pro Gln Glu Thr Thr Val 20 25 30 Lys Leu Ser Cys Asp GluGly Pro Leu Gln Val Ile Leu Gly Pro Glu 35 40 45 Gln Ala Val Val Leu AspCys Thr Leu Gly Ala Thr Ala Ala Gly Pro 50 55 60 Pro Thr Arg Val Thr TrpSer Lys Asp Gly Asp Thr Val Leu Glu His 65 70 75 80 Glu Asn Leu His LeuLeu Pro Asn Gly Ser Leu Trp Leu Ser Ser Pro 85 90 95 Leu Glu Gln Glu AspSer Asp Asp Glu Glu Ala Leu Arg Ile Trp Lys 100 105 110 Val Thr Glu GlySer Tyr Ser Cys Leu Ala His Ser Pro Leu Gly Val 115 120 125 Val Ala SerGln Val Ala Val Val Lys Leu Ala Thr Leu Glu Asp Phe 130 135 140 Ser LeuHis Pro Glu Ser Gln Ile Val Glu Glu Asn Gly Thr Ala Arg 145 150 155 160Phe Glu Cys His Thr Lys Gly Leu Pro Ala Pro Ile Ile Thr Trp Glu 165 170175 Lys Asp Gln Val Thr Val Pro Glu Glu Pro Arg Leu Ile Thr Leu Pro 180185 190 Lys Trp Leu Leu Gln Ile Leu Asp Val Gln Asp Ser Asp Ala Gly Ser195 200 205 Tyr Arg Cys Val Ala Thr Asn Ser Ala Arg Gln Arg Phe Ser GlnGlu 210 215 220 Ala Ser Leu Thr Val Ala Leu Arg Gly Ser Leu Glu Ala ThrArg Gly 225 230 235 240 Gln Asp Val Val Ile Val Ala Ala Pro Glu Asn ThrThr Val Val Ser 245 250 255 Gly Gln Asn Val Val Met Glu Cys Val Ala SerAla Asp Pro Thr Pro 260 265 270 Phe Val Ser Trp Val Arg Gln Asp Gly LysPro Ile Ser Thr Asp Val 275 280 285 Ile Val Leu Gly Arg Thr Asn Leu LeuIle Ala Ser Ala Gln Pro Arg 290 295 300 His Ser Gly Val Tyr Val Cys ArgAla Asn Lys Pro Leu Thr Arg Asp 305 310 315 320 Phe Ala Thr Ala Ala AlaGlu Leu Arg Val Leu Ala Ala Pro Ala Ile 325 330 335 Ser Gln Ala Pro GluAla Leu Ser Arg Thr Arg Ala Ser Thr Ala Arg 340 345 350 Phe Val Cys ArgAla Ser Gly Glu Pro Arg Pro Ala Leu His Trp Leu 355 360 365 His Asp GlyIle Pro Leu Arg Pro Asn Gly Arg Val Lys Val Gln Gly 370 375 380 Gly GlyGly Ser Leu Val Ile Thr Gln Ile Gly Leu Gln Asp Ala Gly 385 390 395 400Tyr Tyr Gln Cys Val Ala Glu Asn Ser Ala Gly Thr Ala Cys Ala Ala 405 410415 Ala Pro Leu Ala Val Val Val Arg Glu Gly Leu Pro Ser Ala Pro Thr 420425 430 Arg Val Thr Ala Thr Pro Leu Ser Ser Ser Ser Val Leu Val Ala Trp435 440 445 Glu Arg Pro Glu Leu His Ser Glu Gln Ile Ile Gly Phe Ser LeuHis 450 455 460 Tyr Gln Lys Ala Arg Gly Val Asp Asn Val Glu Tyr Gln PheAla Val 465 470 475 480 Asn Asn Asp Thr Thr Glu Leu Gln Val Arg Asp LeuGlu Pro Asn Thr 485 490 495 Asp Tyr Glu Phe Tyr Val Val Ala Tyr Ser GlnLeu Gly Ala Ser Arg 500 505 510 Thr Ser Ser Pro Ala Leu Val His Thr LeuAsp Asp Val Pro Ser Ala 515 520 525 Ala Pro Gln Leu Thr Leu Ser Ser ProAsn Pro Ser Asp Ile Arg Val 530 535 540 Ala Trp Leu Pro Leu Pro Ser SerLeu Ser Asn Gly Gln Val Leu Lys 545 550 555 560 Tyr Lys Ile Glu Tyr GlyLeu Gly Lys Glu Asp Gln Val Phe Ser Thr 565 570 575 Glu Val Pro Gly AsnGlu Thr Gln Leu Thr Leu Asn Ser Leu Gln Pro 580 585 590 Asn Lys Val TyrArg Val Arg Ile Ser Ala Gly Thr Gly Ala Gly Tyr 595 600 605 Gly Val ProSer Gln Trp Met Gln His Arg Thr Pro Gly Val His Asn 610 615 620 Gln SerHis Val Pro Phe Ala Pro Ala Glu Leu Lys Val Arg Ala Lys 625 630 635 640Met Glu Ser Leu Val Val Ser Trp Gln Pro Pro Pro His Pro Thr Gln 645 650655 Ile Ser Gly Tyr Lys Leu Tyr Trp Gly Glu Val Gly Thr Glu Glu Glu 660665 670 Ala Asp Gly Asp Arg Pro Pro Gly Gly Arg Gly Asp Gln Ala Trp Asp675 680 685 Val Gly Pro Val Arg Leu Lys Lys Lys Val Lys Gln Tyr Glu LeuThr 690 695 700 Gln Leu Val Pro Gly Arg Pro Tyr Glu Val Lys Leu Val AlaPhe Asn 705 710 715 720 Lys His Glu Asp Gly Tyr Ala Ala Val Trp Lys GlyLys Thr Glu Lys 725 730 735 Ala Pro Thr Pro Asp Leu Pro Ile Gln Arg GlyPro Pro Leu Pro Pro 740 745 750 Ala His Val His Ala Glu Ser Asn Ser SerThr Ser Ile Trp Leu Arg 755 760 765 Trp Lys Lys Pro Asp Phe Thr Thr ValLys Ile Val Asn Tyr Thr Val 770 775 780 Arg Phe Gly Pro Trp Gly Leu ArgAsn Ala Ser Leu Val Thr Tyr Tyr 785 790 795 800 Thr Ser Ser Gly Glu AspIle Leu Ile Gly Gly Leu Lys Pro Phe Thr 805 810 815 Lys Tyr Glu Phe AlaVal Gln Ser His Gly Val Asp Met Asp Gly Pro 820 825 830 Phe Gly Ser ValVal Glu Arg Ser Thr Leu Pro Asp Arg Pro Ser Thr 835 840 845 Pro Pro SerAsp Leu Arg Leu Ser Pro Leu Thr Pro Ser Thr Val Arg 850 855 860 Leu HisTrp Cys Pro Pro Thr Glu Pro Asn Gly Glu Ile Val Glu Tyr 865 870 875 880Leu Ile Leu Tyr Ser Asn Asn His Thr Gln Pro Glu His Gln Trp Thr 885 890895 Leu Leu Thr Thr Glu Gly Asn Ile Phe Ser Ala Glu Val His Gly Leu 900905 910 Glu Ser Asp Thr Arg Tyr Phe Phe Lys Met Gly Ala Arg Thr Glu Val915 920 925 Gly Pro Gly Pro Phe Ser Arg Leu Gln Asp Val Ile Thr Leu GlnGlu 930 935 940 Thr Phe Ser Asp Ser Leu Asp Val His Ala Val Thr Gly IleIle Val 945 950 955 960 Gly Val Cys Leu Gly Leu Leu Cys Leu Leu Ala CysMet Cys Ala Gly 965 970 975 Leu Arg Gln Ser Ser His Arg Glu Ala Leu ProGly Leu Ser Ser Ser 980 985 990 Gly Thr Pro Gly Asn Pro Ala Leu Tyr ThrArg Ala Arg Leu Gly Pro 995 1000 1005 Pro Ser Val Pro Ala Ala His GluLeu Glu Ser Leu Val His Pro Arg 1010 1015 1020 Pro Gln Asp Trp Ser ProPro Pro Ser Asp Val Glu Asp Lys Ala Glu 1025 1030 1035 1040 Val His SerLeu Met Gly Gly Ser Val Ser Asp Cys Arg Gly His Ser 1045 1050 1055 LysArg Lys Ile Ser Trp Ala Gln Ala Gly Gly Pro Asn Trp Ala Gly 1060 10651070 Ser Trp Ala Gly Cys Glu Leu Pro Gln Gly Ser Gly Pro Arg Pro Ala1075 1080 1085 Leu Thr Arg Ala Leu Leu Pro Pro Ala Gly Thr Gly Gln ThrLeu Leu 1090 1095 1100 Leu Gln Ala Leu Val Tyr Asp Gly Ile Lys Ser AsnGly Arg Lys Lys 1105 1110 1115 1120 Pro Ser Pro Ala Cys Arg Asn Gln ValGlu Ala Glu Val Ile Val His 1125 1130 1135 Ser Asp Phe Gly Ala Ser LysGly Cys Pro Asp Leu His Leu Gln Asp 1140 1145 1150 Leu Glu Pro Glu GluPro Leu Thr Ala Glu Thr Leu Pro Ser Thr Ser 1155 1160 1165 Gly Ala ValAsp Leu Ser Gln Gly Ala Asp Trp Leu Gly Arg Glu Leu 1170 1175 1180 GlyGly Cys Gln Pro Thr Thr Ser Gly Pro Glu Arg Leu Thr Cys Leu 1185 11901195 1200 Pro Glu Ala Ala Ser Ala Ser Cys Ser Cys Ser Asp Leu Gln ProSer 1205 1210 1215 Thr Ala Ile Glu Glu Ala Pro Gly Lys Ser Cys Gln ProLys Ala Leu 1220 1225 1230 Cys Pro Leu Thr Val Ser Pro Ser Leu Pro ArgAla Pro Val Ser Ser 1235 1240 1245 Ala Gln Val Pro 1250 3 2796 DNA Musmusculus CDS (1)...(2796) 3 ggg gag ctg cca ttg ccc cag gag aca act gtcaag ctg agc tgt gat 48 Gly Glu Leu Pro Leu Pro Gln Glu Thr Thr Val LysLeu Ser Cys Asp 1 5 10 15 gag gga ccc ctg caa gtg atc ctg ggc cct gagcag gct gtg gtg ctg 96 Glu Gly Pro Leu Gln Val Ile Leu Gly Pro Glu GlnAla Val Val Leu 20 25 30 gac tgc act ttg ggg gct aca gct gct ggg cct ccgacc agg gtg aca 144 Asp Cys Thr Leu Gly Ala Thr Ala Ala Gly Pro Pro ThrArg Val Thr 35 40 45 tgg agc aag gat gga gac act gta cta gag cat gag aacctg cac ctg 192 Trp Ser Lys Asp Gly Asp Thr Val Leu Glu His Glu Asn LeuHis Leu 50 55 60 cta ccc aat ggc tcc ctg tgg ctg tcc tca ccc cta gag caagaa gac 240 Leu Pro Asn Gly Ser Leu Trp Leu Ser Ser Pro Leu Glu Gln GluAsp 65 70 75 80 agc gat gat gag gaa gct ctt agg atc tgg aag gtc act gagggc agc 288 Ser Asp Asp Glu Glu Ala Leu Arg Ile Trp Lys Val Thr Glu GlySer 85 90 95 tat tcc tgt ctg gcc cac agc ccg cta gga gtg gtg gcc agc caggtt 336 Tyr Ser Cys Leu Ala His Ser Pro Leu Gly Val Val Ala Ser Gln Val100 105 110 gct gtg gtc aag ctt gcc aca ctc gaa gac ttc tct ctg cac cccgag 384 Ala Val Val Lys Leu Ala Thr Leu Glu Asp Phe Ser Leu His Pro Glu115 120 125 tcc cag att gtg gag gag aac ggg aca gca cgc ttt gaa tgc cacacc 432 Ser Gln Ile Val Glu Glu Asn Gly Thr Ala Arg Phe Glu Cys His Thr130 135 140 aag ggc ctt cca gcc ccc atc att act tgg gaa aag gac cag gtgacc 480 Lys Gly Leu Pro Ala Pro Ile Ile Thr Trp Glu Lys Asp Gln Val Thr145 150 155 160 gtg cct gag gag ccc cgg ctc atc act ctt ccc aag tgg ctcctc cag 528 Val Pro Glu Glu Pro Arg Leu Ile Thr Leu Pro Lys Trp Leu LeuGln 165 170 175 atc cta gat gtc cag gac agt gat gca ggc tcc tac cgc tgcgtg gcc 576 Ile Leu Asp Val Gln Asp Ser Asp Ala Gly Ser Tyr Arg Cys ValAla 180 185 190 acc aat tca gcc cgc caa cga ttc agc cag gag gcc tcg ctcact gtg 624 Thr Asn Ser Ala Arg Gln Arg Phe Ser Gln Glu Ala Ser Leu ThrVal 195 200 205 gcc ctc aga ggg tct ttg gag gct acc agg ggg cag gat gtggtc att 672 Ala Leu Arg Gly Ser Leu Glu Ala Thr Arg Gly Gln Asp Val ValIle 210 215 220 gtg gca gcc cca gag aac acc acg gta gtg tct gga cag aatgta gtg 720 Val Ala Ala Pro Glu Asn Thr Thr Val Val Ser Gly Gln Asn ValVal 225 230 235 240 atg gag tgc gtg gcc tct gct gac ccc acc cct ttt gtgtcc tgg gtc 768 Met Glu Cys Val Ala Ser Ala Asp Pro Thr Pro Phe Val SerTrp Val 245 250 255 cga cag gat gga aag cct atc tcc acg gat gtc atc gttctg ggc cgg 816 Arg Gln Asp Gly Lys Pro Ile Ser Thr Asp Val Ile Val LeuGly Arg 260 265 270 acc aat cta ctc atc gcc agc gcg cag cct cgg cac tctgga gtc tat 864 Thr Asn Leu Leu Ile Ala Ser Ala Gln Pro Arg His Ser GlyVal Tyr 275 280 285 gtc tgc cga gcc aac aag ccc ctc acg cgt gac ttc gccact gcg gct 912 Val Cys Arg Ala Asn Lys Pro Leu Thr Arg Asp Phe Ala ThrAla Ala 290 295 300 gct gag ctc cga gtg ctt gct gcc cca gcc atc tcg caggca ccc gag 960 Ala Glu Leu Arg Val Leu Ala Ala Pro Ala Ile Ser Gln AlaPro Glu 305 310 315 320 gcg ctc tcg cgg acg cgg gcc agc acc gcg cgc ttcgtg tgc cgg gcg 1008 Ala Leu Ser Arg Thr Arg Ala Ser Thr Ala Arg Phe ValCys Arg Ala 325 330 335 tcc ggg gag cca cgg ccc gcg ctg cac tgg ctg cacgac ggg atc ccg 1056 Ser Gly Glu Pro Arg Pro Ala Leu His Trp Leu His AspGly Ile Pro 340 345 350 ttg cga ccc aat ggg cgc gtc aag gtg cag ggc ggtggc ggc agc ttg 1104 Leu Arg Pro Asn Gly Arg Val Lys Val Gln Gly Gly GlyGly Ser Leu 355 360 365 gtc atc act cag atc ggc ctg cag gac gct ggc tactac cag tgc gta 1152 Val Ile Thr Gln Ile Gly Leu Gln Asp Ala Gly Tyr TyrGln Cys Val 370 375 380 gca gaa aac agc gcg gga act gcc tgt gcc gct gcgccc ctg gcg gta 1200 Ala Glu Asn Ser Ala Gly Thr Ala Cys Ala Ala Ala ProLeu Ala Val 385 390 395 400 gtg gtg cgc gag ggg ctg ccc agc gcc ccg actcgg gtc aca gcc acg 1248 Val Val Arg Glu Gly Leu Pro Ser Ala Pro Thr ArgVal Thr Ala Thr 405 410 415 ccg ctg agc agc tcc tct gtg ctg gtg gcc tgggag cgg cct gag ttg 1296 Pro Leu Ser Ser Ser Ser Val Leu Val Ala Trp GluArg Pro Glu Leu 420 425 430 cac agc gag caa atc att ggc ttc tct ctt cactac caa aag gca agg 1344 His Ser Glu Gln Ile Ile Gly Phe Ser Leu His TyrGln Lys Ala Arg 435 440 445 gga gtg gac aat gtg gag tac cag ttt gca gtaaac aat gac acc aca 1392 Gly Val Asp Asn Val Glu Tyr Gln Phe Ala Val AsnAsn Asp Thr Thr 450 455 460 gag ctg cag gtt cgg gac ctg gaa ccc aac acggat tat gag ttc tac 1440 Glu Leu Gln Val Arg Asp Leu Glu Pro Asn Thr AspTyr Glu Phe Tyr 465 470 475 480 gtg gtg gcc tac tcc cag ctg ggg gcc agccga acc tcc agc cca gcc 1488 Val Val Ala Tyr Ser Gln Leu Gly Ala Ser ArgThr Ser Ser Pro Ala 485 490 495 ctg gtg cat aca ctg gac gat gtc ccc agcgca gca ccc cag ctt acc 1536 Leu Val His Thr Leu Asp Asp Val Pro Ser AlaAla Pro Gln Leu Thr 500 505 510 ttg tcc agc ccc aac ccc tcg gac atc agggtg gca tgg ctg ccc ctg 1584 Leu Ser Ser Pro Asn Pro Ser Asp Ile Arg ValAla Trp Leu Pro Leu 515 520 525 ccc tcc agc ctg agc aat gga cag gtg ctgaag tac aag ata gag tac 1632 Pro Ser Ser Leu Ser Asn Gly Gln Val Leu LysTyr Lys Ile Glu Tyr 530 535 540 ggt ttg ggg aag gaa gat cag gtt ttc tccacc gag gtg cct gga aat 1680 Gly Leu Gly Lys Glu Asp Gln Val Phe Ser ThrGlu Val Pro Gly Asn 545 550 555 560 gag aca caa ctt acg tta aac tca cttcag cca aac aaa gtg tac cga 1728 Glu Thr Gln Leu Thr Leu Asn Ser Leu GlnPro Asn Lys Val Tyr Arg 565 570 575 gtc cgg att tca gct ggc act ggc gctggc tat gga gtc cct tct cag 1776 Val Arg Ile Ser Ala Gly Thr Gly Ala GlyTyr Gly Val Pro Ser Gln 580 585 590 tgg atg cag cac agg aca cct ggt gtgcac aac cag agc cat gtt ccc 1824 Trp Met Gln His Arg Thr Pro Gly Val HisAsn Gln Ser His Val Pro 595 600 605 ttt gcc cct gca gaa ttg aag gtg agggca aag atg gag tcc ctg gtg 1872 Phe Ala Pro Ala Glu Leu Lys Val Arg AlaLys Met Glu Ser Leu Val 610 615 620 gtg tca tgg cag ccg ccc cct cac cccacc cag atc tct gga tac aaa 1920 Val Ser Trp Gln Pro Pro Pro His Pro ThrGln Ile Ser Gly Tyr Lys 625 630 635 640 ctc tac tgg gga gag gtg gga acagag gag gag gca gat ggt gac cgc 1968 Leu Tyr Trp Gly Glu Val Gly Thr GluGlu Glu Ala Asp Gly Asp Arg 645 650 655 ccc cca ggg ggt cgt gga gat caagct tgg gac gtc ggg ccc gtg cgg 2016 Pro Pro Gly Gly Arg Gly Asp Gln AlaTrp Asp Val Gly Pro Val Arg 660 665 670 ctg aag aag aaa gtg aag cag tatgaa ctg acc cag tta gtc cct ggc 2064 Leu Lys Lys Lys Val Lys Gln Tyr GluLeu Thr Gln Leu Val Pro Gly 675 680 685 agg ccg tac gag gtg aag ctc gtagct ttc aac aaa cac gag gac ggc 2112 Arg Pro Tyr Glu Val Lys Leu Val AlaPhe Asn Lys His Glu Asp Gly 690 695 700 tac gct gct gtg tgg aag ggc aagacg gag aag gcg ccc acg cca gac 2160 Tyr Ala Ala Val Trp Lys Gly Lys ThrGlu Lys Ala Pro Thr Pro Asp 705 710 715 720 ctg cct atc cag agg ggg ccaccg ctg cct cct gcc cat gtc cac gca 2208 Leu Pro Ile Gln Arg Gly Pro ProLeu Pro Pro Ala His Val His Ala 725 730 735 gag tca aac agc tcc act tccatt tgg ctt cgg tgg aag aag cca gac 2256 Glu Ser Asn Ser Ser Thr Ser IleTrp Leu Arg Trp Lys Lys Pro Asp 740 745 750 ttt acc act gtc aag att gtcaac tac act gta cgc ttc ggc ccc tgg 2304 Phe Thr Thr Val Lys Ile Val AsnTyr Thr Val Arg Phe Gly Pro Trp 755 760 765 ggg ctc agg aat gct tcc ctggtc acc tac tat acc agc tct gga gaa 2352 Gly Leu Arg Asn Ala Ser Leu ValThr Tyr Tyr Thr Ser Ser Gly Glu 770 775 780 gac att ctc att ggc ggc ctgaaa cca ttt acc aag tac gag ttt gcg 2400 Asp Ile Leu Ile Gly Gly Leu LysPro Phe Thr Lys Tyr Glu Phe Ala 785 790 795 800 gta cag tcc cac gga gtggat atg gat ggg ccc ttt ggc tcc gtc gta 2448 Val Gln Ser His Gly Val AspMet Asp Gly Pro Phe Gly Ser Val Val 805 810 815 gaa cgc tcc acc ctg cctgac cgg cct tca aca cct cct tct gac ctg 2496 Glu Arg Ser Thr Leu Pro AspArg Pro Ser Thr Pro Pro Ser Asp Leu 820 825 830 cgc ctg agc ccc ctg acacca tcc acc gtt cgg tta cac tgg tgt ccc 2544 Arg Leu Ser Pro Leu Thr ProSer Thr Val Arg Leu His Trp Cys Pro 835 840 845 ccc acg gag ccc aat ggtgag att gtg gag tat cta att ctc tac agc 2592 Pro Thr Glu Pro Asn Gly GluIle Val Glu Tyr Leu Ile Leu Tyr Ser 850 855 860 aac aac cac acc cag cccgaa cac cag tgg aca ctg ctc acc aca gag 2640 Asn Asn His Thr Gln Pro GluHis Gln Trp Thr Leu Leu Thr Thr Glu 865 870 875 880 gga aac atc ttc agtgca gag gtc cat ggc cta gag agt gac act cgg 2688 Gly Asn Ile Phe Ser AlaGlu Val His Gly Leu Glu Ser Asp Thr Arg 885 890 895 tat ttc ttc aag atggga gcc cgc aca gag gtg ggg cct ggg ccc ttt 2736 Tyr Phe Phe Lys Met GlyAla Arg Thr Glu Val Gly Pro Gly Pro Phe 900 905 910 tcc cgc ttg cag gatgtg att act ctg caa gag aca ttc tca gac tcc 2784 Ser Arg Leu Gln Asp ValIle Thr Leu Gln Glu Thr Phe Ser Asp Ser 915 920 925 ttg gat gtg cac 2796Leu Asp Val His 930 4 932 PRT Mus musculus 4 Gly Glu Leu Pro Leu Pro GlnGlu Thr Thr Val Lys Leu Ser Cys Asp 1 5 10 15 Glu Gly Pro Leu Gln ValIle Leu Gly Pro Glu Gln Ala Val Val Leu 20 25 30 Asp Cys Thr Leu Gly AlaThr Ala Ala Gly Pro Pro Thr Arg Val Thr 35 40 45 Trp Ser Lys Asp Gly AspThr Val Leu Glu His Glu Asn Leu His Leu 50 55 60 Leu Pro Asn Gly Ser LeuTrp Leu Ser Ser Pro Leu Glu Gln Glu Asp 65 70 75 80 Ser Asp Asp Glu GluAla Leu Arg Ile Trp Lys Val Thr Glu Gly Ser 85 90 95 Tyr Ser Cys Leu AlaHis Ser Pro Leu Gly Val Val Ala Ser Gln Val 100 105 110 Ala Val Val LysLeu Ala Thr Leu Glu Asp Phe Ser Leu His Pro Glu 115 120 125 Ser Gln IleVal Glu Glu Asn Gly Thr Ala Arg Phe Glu Cys His Thr 130 135 140 Lys GlyLeu Pro Ala Pro Ile Ile Thr Trp Glu Lys Asp Gln Val Thr 145 150 155 160Val Pro Glu Glu Pro Arg Leu Ile Thr Leu Pro Lys Trp Leu Leu Gln 165 170175 Ile Leu Asp Val Gln Asp Ser Asp Ala Gly Ser Tyr Arg Cys Val Ala 180185 190 Thr Asn Ser Ala Arg Gln Arg Phe Ser Gln Glu Ala Ser Leu Thr Val195 200 205 Ala Leu Arg Gly Ser Leu Glu Ala Thr Arg Gly Gln Asp Val ValIle 210 215 220 Val Ala Ala Pro Glu Asn Thr Thr Val Val Ser Gly Gln AsnVal Val 225 230 235 240 Met Glu Cys Val Ala Ser Ala Asp Pro Thr Pro PheVal Ser Trp Val 245 250 255 Arg Gln Asp Gly Lys Pro Ile Ser Thr Asp ValIle Val Leu Gly Arg 260 265 270 Thr Asn Leu Leu Ile Ala Ser Ala Gln ProArg His Ser Gly Val Tyr 275 280 285 Val Cys Arg Ala Asn Lys Pro Leu ThrArg Asp Phe Ala Thr Ala Ala 290 295 300 Ala Glu Leu Arg Val Leu Ala AlaPro Ala Ile Ser Gln Ala Pro Glu 305 310 315 320 Ala Leu Ser Arg Thr ArgAla Ser Thr Ala Arg Phe Val Cys Arg Ala 325 330 335 Ser Gly Glu Pro ArgPro Ala Leu His Trp Leu His Asp Gly Ile Pro 340 345 350 Leu Arg Pro AsnGly Arg Val Lys Val Gln Gly Gly Gly Gly Ser Leu 355 360 365 Val Ile ThrGln Ile Gly Leu Gln Asp Ala Gly Tyr Tyr Gln Cys Val 370 375 380 Ala GluAsn Ser Ala Gly Thr Ala Cys Ala Ala Ala Pro Leu Ala Val 385 390 395 400Val Val Arg Glu Gly Leu Pro Ser Ala Pro Thr Arg Val Thr Ala Thr 405 410415 Pro Leu Ser Ser Ser Ser Val Leu Val Ala Trp Glu Arg Pro Glu Leu 420425 430 His Ser Glu Gln Ile Ile Gly Phe Ser Leu His Tyr Gln Lys Ala Arg435 440 445 Gly Val Asp Asn Val Glu Tyr Gln Phe Ala Val Asn Asn Asp ThrThr 450 455 460 Glu Leu Gln Val Arg Asp Leu Glu Pro Asn Thr Asp Tyr GluPhe Tyr 465 470 475 480 Val Val Ala Tyr Ser Gln Leu Gly Ala Ser Arg ThrSer Ser Pro Ala 485 490 495 Leu Val His Thr Leu Asp Asp Val Pro Ser AlaAla Pro Gln Leu Thr 500 505 510 Leu Ser Ser Pro Asn Pro Ser Asp Ile ArgVal Ala Trp Leu Pro Leu 515 520 525 Pro Ser Ser Leu Ser Asn Gly Gln ValLeu Lys Tyr Lys Ile Glu Tyr 530 535 540 Gly Leu Gly Lys Glu Asp Gln ValPhe Ser Thr Glu Val Pro Gly Asn 545 550 555 560 Glu Thr Gln Leu Thr LeuAsn Ser Leu Gln Pro Asn Lys Val Tyr Arg 565 570 575 Val Arg Ile Ser AlaGly Thr Gly Ala Gly Tyr Gly Val Pro Ser Gln 580 585 590 Trp Met Gln HisArg Thr Pro Gly Val His Asn Gln Ser His Val Pro 595 600 605 Phe Ala ProAla Glu Leu Lys Val Arg Ala Lys Met Glu Ser Leu Val 610 615 620 Val SerTrp Gln Pro Pro Pro His Pro Thr Gln Ile Ser Gly Tyr Lys 625 630 635 640Leu Tyr Trp Gly Glu Val Gly Thr Glu Glu Glu Ala Asp Gly Asp Arg 645 650655 Pro Pro Gly Gly Arg Gly Asp Gln Ala Trp Asp Val Gly Pro Val Arg 660665 670 Leu Lys Lys Lys Val Lys Gln Tyr Glu Leu Thr Gln Leu Val Pro Gly675 680 685 Arg Pro Tyr Glu Val Lys Leu Val Ala Phe Asn Lys His Glu AspGly 690 695 700 Tyr Ala Ala Val Trp Lys Gly Lys Thr Glu Lys Ala Pro ThrPro Asp 705 710 715 720 Leu Pro Ile Gln Arg Gly Pro Pro Leu Pro Pro AlaHis Val His Ala 725 730 735 Glu Ser Asn Ser Ser Thr Ser Ile Trp Leu ArgTrp Lys Lys Pro Asp 740 745 750 Phe Thr Thr Val Lys Ile Val Asn Tyr ThrVal Arg Phe Gly Pro Trp 755 760 765 Gly Leu Arg Asn Ala Ser Leu Val ThrTyr Tyr Thr Ser Ser Gly Glu 770 775 780 Asp Ile Leu Ile Gly Gly Leu LysPro Phe Thr Lys Tyr Glu Phe Ala 785 790 795 800 Val Gln Ser His Gly ValAsp Met Asp Gly Pro Phe Gly Ser Val Val 805 810 815 Glu Arg Ser Thr LeuPro Asp Arg Pro Ser Thr Pro Pro Ser Asp Leu 820 825 830 Arg Leu Ser ProLeu Thr Pro Ser Thr Val Arg Leu His Trp Cys Pro 835 840 845 Pro Thr GluPro Asn Gly Glu Ile Val Glu Tyr Leu Ile Leu Tyr Ser 850 855 860 Asn AsnHis Thr Gln Pro Glu His Gln Trp Thr Leu Leu Thr Thr Glu 865 870 875 880Gly Asn Ile Phe Ser Ala Glu Val His Gly Leu Glu Ser Asp Thr Arg 885 890895 Tyr Phe Phe Lys Met Gly Ala Arg Thr Glu Val Gly Pro Gly Pro Phe 900905 910 Ser Arg Leu Gln Asp Val Ile Thr Leu Gln Glu Thr Phe Ser Asp Ser915 920 925 Leu Asp Val His 930 5 825 DNA Mus musculus CDS (1)...(825) 5cga caa agc tcc cac agg gaa gcc ctt ccc gga ttg tcc tcc tca ggc 48 ArgGln Ser Ser His Arg Glu Ala Leu Pro Gly Leu Ser Ser Ser Gly 1 5 10 15acc cca gga aac cca gcg ctc tac aca aga gct cgg ctt ggg cct ccc 96 ThrPro Gly Asn Pro Ala Leu Tyr Thr Arg Ala Arg Leu Gly Pro Pro 20 25 30 agtgtc cct gct gcc cat gag ttg gag tcc ctc gtg cat cct cgt ccc 144 Ser ValPro Ala Ala His Glu Leu Glu Ser Leu Val His Pro Arg Pro 35 40 45 cag gattgg tcc cca cca ccc tca gat gtg gaa gac aag gct gaa gta 192 Gln Asp TrpSer Pro Pro Pro Ser Asp Val Glu Asp Lys Ala Glu Val 50 55 60 cac agc cttatg ggt ggc agt gtt tca gat tgc cgg ggc cac tcc aag 240 His Ser Leu MetGly Gly Ser Val Ser Asp Cys Arg Gly His Ser Lys 65 70 75 80 aga aag atctcc tgg gct cag gca ggg gga cca aac tgg gca ggc tcc 288 Arg Lys Ile SerTrp Ala Gln Ala Gly Gly Pro Asn Trp Ala Gly Ser 85 90 95 tgg gca ggc tgtgag ctg ccc cag ggt agt ggt cca agg ccg gct ctg 336 Trp Ala Gly Cys GluLeu Pro Gln Gly Ser Gly Pro Arg Pro Ala Leu 100 105 110 acc cgt gct ctgctg cct cca gcg gga acc ggg cag aca ctg ctg ctg 384 Thr Arg Ala Leu LeuPro Pro Ala Gly Thr Gly Gln Thr Leu Leu Leu 115 120 125 caa gcc ctg gtgtat gac ggc ata aag agc aac ggg aga aag aag ccg 432 Gln Ala Leu Val TyrAsp Gly Ile Lys Ser Asn Gly Arg Lys Lys Pro 130 135 140 tcc cca gcc tgcagg aat cag gtg gaa gct gag gtc att gtc cac tcc 480 Ser Pro Ala Cys ArgAsn Gln Val Glu Ala Glu Val Ile Val His Ser 145 150 155 160 gac ttc ggtgca tcc aaa gga tgt cct gac ctc cac ctc caa gac ctg 528 Asp Phe Gly AlaSer Lys Gly Cys Pro Asp Leu His Leu Gln Asp Leu 165 170 175 gag cca gaggaa cca ctg act gca gag act ctg cct tcc acg tct gga 576 Glu Pro Glu GluPro Leu Thr Ala Glu Thr Leu Pro Ser Thr Ser Gly 180 185 190 gct gtg gatctg tct caa gga gca gac tgg ctg ggc agg gag ctg gga 624 Ala Val Asp LeuSer Gln Gly Ala Asp Trp Leu Gly Arg Glu Leu Gly 195 200 205 ggg tgc caacca aca acc agt ggg cca gag agg ctc acc tgc ttg cca 672 Gly Cys Gln ProThr Thr Ser Gly Pro Glu Arg Leu Thr Cys Leu Pro 210 215 220 gaa gca gccagt gcc tcc tgc tcc tgc tca gac ctc cag ccc agc act 720 Glu Ala Ala SerAla Ser Cys Ser Cys Ser Asp Leu Gln Pro Ser Thr 225 230 235 240 gct atagag gag gcc cct ggg aaa agc tgc cag ccc aaa gcc ctg tgt 768 Ala Ile GluGlu Ala Pro Gly Lys Ser Cys Gln Pro Lys Ala Leu Cys 245 250 255 cct ctaaca gtc agc cca agc ctt ccc agg gcc cct gtc tcc tct gct 816 Pro Leu ThrVal Ser Pro Ser Leu Pro Arg Ala Pro Val Ser Ser Ala 260 265 270 cag gtcccc 825 Gln Val Pro 275 6 275 PRT Mus musculus 6 Arg Gln Ser Ser His ArgGlu Ala Leu Pro Gly Leu Ser Ser Ser Gly 1 5 10 15 Thr Pro Gly Asn ProAla Leu Tyr Thr Arg Ala Arg Leu Gly Pro Pro 20 25 30 Ser Val Pro Ala AlaHis Glu Leu Glu Ser Leu Val His Pro Arg Pro 35 40 45 Gln Asp Trp Ser ProPro Pro Ser Asp Val Glu Asp Lys Ala Glu Val 50 55 60 His Ser Leu Met GlyGly Ser Val Ser Asp Cys Arg Gly His Ser Lys 65 70 75 80 Arg Lys Ile SerTrp Ala Gln Ala Gly Gly Pro Asn Trp Ala Gly Ser 85 90 95 Trp Ala Gly CysGlu Leu Pro Gln Gly Ser Gly Pro Arg Pro Ala Leu 100 105 110 Thr Arg AlaLeu Leu Pro Pro Ala Gly Thr Gly Gln Thr Leu Leu Leu 115 120 125 Gln AlaLeu Val Tyr Asp Gly Ile Lys Ser Asn Gly Arg Lys Lys Pro 130 135 140 SerPro Ala Cys Arg Asn Gln Val Glu Ala Glu Val Ile Val His Ser 145 150 155160 Asp Phe Gly Ala Ser Lys Gly Cys Pro Asp Leu His Leu Gln Asp Leu 165170 175 Glu Pro Glu Glu Pro Leu Thr Ala Glu Thr Leu Pro Ser Thr Ser Gly180 185 190 Ala Val Asp Leu Ser Gln Gly Ala Asp Trp Leu Gly Arg Glu LeuGly 195 200 205 Gly Cys Gln Pro Thr Thr Ser Gly Pro Glu Arg Leu Thr CysLeu Pro 210 215 220 Glu Ala Ala Ser Ala Ser Cys Ser Cys Ser Asp Leu GlnPro Ser Thr 225 230 235 240 Ala Ile Glu Glu Ala Pro Gly Lys Ser Cys GlnPro Lys Ala Leu Cys 245 250 255 Pro Leu Thr Val Ser Pro Ser Leu Pro ArgAla Pro Val Ser Ser Ala 260 265 270 Gln Val Pro 275 7 243 DNA Musmusculus CDS (1)...(243) 7 cct gag cag gct gtg gtg ctg gac tgc act ttgggg gct aca gct gct 48 Pro Glu Gln Ala Val Val Leu Asp Cys Thr Leu GlyAla Thr Ala Ala 1 5 10 15 ggg cct ccg acc agg gtg aca tgg agc aag gatgga gac act gta cta 96 Gly Pro Pro Thr Arg Val Thr Trp Ser Lys Asp GlyAsp Thr Val Leu 20 25 30 gag cat gag aac ctg cac ctg cta ccc aat ggc tccctg tgg ctg tcc 144 Glu His Glu Asn Leu His Leu Leu Pro Asn Gly Ser LeuTrp Leu Ser 35 40 45 tca ccc cta gag caa gaa gac agc gat gat gag gaa gctctt agg atc 192 Ser Pro Leu Glu Gln Glu Asp Ser Asp Asp Glu Glu Ala LeuArg Ile 50 55 60 tgg aag gtc act gag ggc agc tat tcc tgt ctg gcc cac agcccg cta 240 Trp Lys Val Thr Glu Gly Ser Tyr Ser Cys Leu Ala His Ser ProLeu 65 70 75 80 gga 243 Gly 8 81 PRT Mus musculus 8 Pro Glu Gln Ala ValVal Leu Asp Cys Thr Leu Gly Ala Thr Ala Ala 1 5 10 15 Gly Pro Pro ThrArg Val Thr Trp Ser Lys Asp Gly Asp Thr Val Leu 20 25 30 Glu His Glu AsnLeu His Leu Leu Pro Asn Gly Ser Leu Trp Leu Ser 35 40 45 Ser Pro Leu GluGln Glu Asp Ser Asp Asp Glu Glu Ala Leu Arg Ile 50 55 60 Trp Lys Val ThrGlu Gly Ser Tyr Ser Cys Leu Ala His Ser Pro Leu 65 70 75 80 Gly 9 192DNA Mus musculus CDS (1)...(192) 9 gag aac ggg aca gca cgc ttt gaa tgccac acc aag ggc ctt cca gcc 48 Glu Asn Gly Thr Ala Arg Phe Glu Cys HisThr Lys Gly Leu Pro Ala 1 5 10 15 ccc atc att act tgg gaa aag gac caggtg acc gtg cct gag gag ccc 96 Pro Ile Ile Thr Trp Glu Lys Asp Gln ValThr Val Pro Glu Glu Pro 20 25 30 cgg ctc atc act ctt ccc aag tgg ctc ctccag atc cta gat gtc cag 144 Arg Leu Ile Thr Leu Pro Lys Trp Leu Leu GlnIle Leu Asp Val Gln 35 40 45 gac agt gat gca ggc tcc tac cgc tgc gtg gccacc aat tca gcc cgc 192 Asp Ser Asp Ala Gly Ser Tyr Arg Cys Val Ala ThrAsn Ser Ala Arg 50 55 60 10 64 PRT Mus musculus 10 Glu Asn Gly Thr AlaArg Phe Glu Cys His Thr Lys Gly Leu Pro Ala 1 5 10 15 Pro Ile Ile ThrTrp Glu Lys Asp Gln Val Thr Val Pro Glu Glu Pro 20 25 30 Arg Leu Ile ThrLeu Pro Lys Trp Leu Leu Gln Ile Leu Asp Val Gln 35 40 45 Asp Ser Asp AlaGly Ser Tyr Arg Cys Val Ala Thr Asn Ser Ala Arg 50 55 60 11 189 DNA Musmusculus CDS (1)...(189) 11 tct gga cag aat gta gtg atg gag tgc gtg gcctct gct gac ccc acc 48 Ser Gly Gln Asn Val Val Met Glu Cys Val Ala SerAla Asp Pro Thr 1 5 10 15 cct ttt gtg tcc tgg gtc cga cag gat gga aagcct atc tcc acg gat 96 Pro Phe Val Ser Trp Val Arg Gln Asp Gly Lys ProIle Ser Thr Asp 20 25 30 gtc atc gtt ctg ggc cgg acc aat cta ctc atc gccagc gcg cag cct 144 Val Ile Val Leu Gly Arg Thr Asn Leu Leu Ile Ala SerAla Gln Pro 35 40 45 cgg cac tct gga gtc tat gtc tgc cga gcc aac aag cccctc acg 189 Arg His Ser Gly Val Tyr Val Cys Arg Ala Asn Lys Pro Leu Thr50 55 60 12 63 PRT Mus musculus 12 Ser Gly Gln Asn Val Val Met Glu CysVal Ala Ser Ala Asp Pro Thr 1 5 10 15 Pro Phe Val Ser Trp Val Arg GlnAsp Gly Lys Pro Ile Ser Thr Asp 20 25 30 Val Ile Val Leu Gly Arg Thr AsnLeu Leu Ile Ala Ser Ala Gln Pro 35 40 45 Arg His Ser Gly Val Tyr Val CysArg Ala Asn Lys Pro Leu Thr 50 55 60 13 195 DNA Mus musculus CDS(1)...(195) 13 cgg gcc agc acc gcg cgc ttc gtg tgc cgg gcg tcc ggg gagcca cgg 48 Arg Ala Ser Thr Ala Arg Phe Val Cys Arg Ala Ser Gly Glu ProArg 1 5 10 15 ccc gcg ctg cac tgg ctg cac gac ggg atc ccg ttg cga cccaat ggg 96 Pro Ala Leu His Trp Leu His Asp Gly Ile Pro Leu Arg Pro AsnGly 20 25 30 cgc gtc aag gtg cag ggc ggt ggc ggc agc ttg gtc atc act cagatc 144 Arg Val Lys Val Gln Gly Gly Gly Gly Ser Leu Val Ile Thr Gln Ile35 40 45 ggc ctg cag gac gct ggc tac tac cag tgc gta gca gaa aac agc gcg192 Gly Leu Gln Asp Ala Gly Tyr Tyr Gln Cys Val Ala Glu Asn Ser Ala 5055 60 gga 195 Gly 65 14 65 PRT Mus musculus 14 Arg Ala Ser Thr Ala ArgPhe Val Cys Arg Ala Ser Gly Glu Pro Arg 1 5 10 15 Pro Ala Leu His TrpLeu His Asp Gly Ile Pro Leu Arg Pro Asn Gly 20 25 30 Arg Val Lys Val GlnGly Gly Gly Gly Ser Leu Val Ile Thr Gln Ile 35 40 45 Gly Leu Gln Asp AlaGly Tyr Tyr Gln Cys Val Ala Glu Asn Ser Ala 50 55 60 Gly 65 15 249 DNAMus musculus CDS (1)...(249) 15 agc gcc ccg act cgg gtc aca gcc acg ccgctg agc agc tcc tct gtg 48 Ser Ala Pro Thr Arg Val Thr Ala Thr Pro LeuSer Ser Ser Ser Val 1 5 10 15 ctg gtg gcc tgg gag cgg cct gag ttg cacagc gag caa atc att ggc 96 Leu Val Ala Trp Glu Arg Pro Glu Leu His SerGlu Gln Ile Ile Gly 20 25 30 ttc tct ctt cac tac caa aag gca agg gga gtggac aat gtg gag tac 144 Phe Ser Leu His Tyr Gln Lys Ala Arg Gly Val AspAsn Val Glu Tyr 35 40 45 cag ttt gca gta aac aat gac acc aca gag ctg caggtt cgg gac ctg 192 Gln Phe Ala Val Asn Asn Asp Thr Thr Glu Leu Gln ValArg Asp Leu 50 55 60 gaa ccc aac acg gat tat gag ttc tac gtg gtg gcc tactcc cag ctg 240 Glu Pro Asn Thr Asp Tyr Glu Phe Tyr Val Val Ala Tyr SerGln Leu 65 70 75 80 ggg gcc agc 249 Gly Ala Ser 16 83 PRT Mus musculus16 Ser Ala Pro Thr Arg Val Thr Ala Thr Pro Leu Ser Ser Ser Ser Val 1 510 15 Leu Val Ala Trp Glu Arg Pro Glu Leu His Ser Glu Gln Ile Ile Gly 2025 30 Phe Ser Leu His Tyr Gln Lys Ala Arg Gly Val Asp Asn Val Glu Tyr 3540 45 Gln Phe Ala Val Asn Asn Asp Thr Thr Glu Leu Gln Val Arg Asp Leu 5055 60 Glu Pro Asn Thr Asp Tyr Glu Phe Tyr Val Val Ala Tyr Ser Gln Leu 6570 75 80 Gly Ala Ser 17 249 DNA Mus musculus CDS (1)...(249) 17 agc gcagca ccc cag ctt acc ttg tcc agc ccc aac ccc tcg gac atc 48 Ser Ala AlaPro Gln Leu Thr Leu Ser Ser Pro Asn Pro Ser Asp Ile 1 5 10 15 agg gtggca tgg ctg ccc ctg ccc tcc agc ctg agc aat gga cag gtg 96 Arg Val AlaTrp Leu Pro Leu Pro Ser Ser Leu Ser Asn Gly Gln Val 20 25 30 ctg aag tacaag ata gag tac ggt ttg ggg aag gaa gat cag gtt ttc 144 Leu Lys Tyr LysIle Glu Tyr Gly Leu Gly Lys Glu Asp Gln Val Phe 35 40 45 tcc acc gag gtgcct gga aat gag aca caa ctt acg tta aac tca ctt 192 Ser Thr Glu Val ProGly Asn Glu Thr Gln Leu Thr Leu Asn Ser Leu 50 55 60 cag cca aac aaa gtgtac cga gtc cgg att tca gct ggc act ggc gct 240 Gln Pro Asn Lys Val TyrArg Val Arg Ile Ser Ala Gly Thr Gly Ala 65 70 75 80 ggc tat gga 249 GlyTyr Gly 18 83 PRT Mus musculus 18 Ser Ala Ala Pro Gln Leu Thr Leu SerSer Pro Asn Pro Ser Asp Ile 1 5 10 15 Arg Val Ala Trp Leu Pro Leu ProSer Ser Leu Ser Asn Gly Gln Val 20 25 30 Leu Lys Tyr Lys Ile Glu Tyr GlyLeu Gly Lys Glu Asp Gln Val Phe 35 40 45 Ser Thr Glu Val Pro Gly Asn GluThr Gln Leu Thr Leu Asn Ser Leu 50 55 60 Gln Pro Asn Lys Val Tyr Arg ValArg Ile Ser Ala Gly Thr Gly Ala 65 70 75 80 Gly Tyr Gly 19 288 DNA Musmusculus CDS (1)...(288) 19 ttt gcc cct gca gaa ttg aag gtg agg gca aagatg gag tcc ctg gtg 48 Phe Ala Pro Ala Glu Leu Lys Val Arg Ala Lys MetGlu Ser Leu Val 1 5 10 15 gtg tca tgg cag ccg ccc cct cac ccc acc cagatc tct gga tac aaa 96 Val Ser Trp Gln Pro Pro Pro His Pro Thr Gln IleSer Gly Tyr Lys 20 25 30 ctc tac tgg gga gag gtg gga aca gag gag gag gcagat ggt gac cgc 144 Leu Tyr Trp Gly Glu Val Gly Thr Glu Glu Glu Ala AspGly Asp Arg 35 40 45 ccc cca ggg ggt cgt gga gat caa gct tgg gac gtc gggccc gtg cgg 192 Pro Pro Gly Gly Arg Gly Asp Gln Ala Trp Asp Val Gly ProVal Arg 50 55 60 ctg aag aag aaa gtg aag cag tat gaa ctg acc cag tta gtccct ggc 240 Leu Lys Lys Lys Val Lys Gln Tyr Glu Leu Thr Gln Leu Val ProGly 65 70 75 80 agg ccg tac gag gtg aag ctc gta gct ttc aac aaa cac gaggac ggc 288 Arg Pro Tyr Glu Val Lys Leu Val Ala Phe Asn Lys His Glu AspGly 85 90 95 20 96 PRT Mus musculus 20 Phe Ala Pro Ala Glu Leu Lys ValArg Ala Lys Met Glu Ser Leu Val 1 5 10 15 Val Ser Trp Gln Pro Pro ProHis Pro Thr Gln Ile Ser Gly Tyr Lys 20 25 30 Leu Tyr Trp Gly Glu Val GlyThr Glu Glu Glu Ala Asp Gly Asp Arg 35 40 45 Pro Pro Gly Gly Arg Gly AspGln Ala Trp Asp Val Gly Pro Val Arg 50 55 60 Leu Lys Lys Lys Val Lys GlnTyr Glu Leu Thr Gln Leu Val Pro Gly 65 70 75 80 Arg Pro Tyr Glu Val LysLeu Val Ala Phe Asn Lys His Glu Asp Gly 85 90 95 21 246 DNA Mus musculusCDS (1)...(246) 21 ctg cct cct gcc cat gtc cac gca gag tca aac agc tccact tcc att 48 Leu Pro Pro Ala His Val His Ala Glu Ser Asn Ser Ser ThrSer Ile 1 5 10 15 tgg ctt cgg tgg aag aag cca gac ttt acc act gtc aagatt gtc aac 96 Trp Leu Arg Trp Lys Lys Pro Asp Phe Thr Thr Val Lys IleVal Asn 20 25 30 tac act gta cgc ttc ggc ccc tgg ggg ctc agg aat gct tccctg gtc 144 Tyr Thr Val Arg Phe Gly Pro Trp Gly Leu Arg Asn Ala Ser LeuVal 35 40 45 acc tac tat acc agc tct gga gaa gac att ctc att ggc ggc ctgaaa 192 Thr Tyr Tyr Thr Ser Ser Gly Glu Asp Ile Leu Ile Gly Gly Leu Lys50 55 60 cca ttt acc aag tac gag ttt gcg gta cag tcc cac gga gtg gat atg240 Pro Phe Thr Lys Tyr Glu Phe Ala Val Gln Ser His Gly Val Asp Met 6570 75 80 gat ggg 246 Asp Gly 22 82 PRT Mus musculus 22 Leu Pro Pro AlaHis Val His Ala Glu Ser Asn Ser Ser Thr Ser Ile 1 5 10 15 Trp Leu ArgTrp Lys Lys Pro Asp Phe Thr Thr Val Lys Ile Val Asn 20 25 30 Tyr Thr ValArg Phe Gly Pro Trp Gly Leu Arg Asn Ala Ser Leu Val 35 40 45 Thr Tyr TyrThr Ser Ser Gly Glu Asp Ile Leu Ile Gly Gly Leu Lys 50 55 60 Pro Phe ThrLys Tyr Glu Phe Ala Val Gln Ser His Gly Val Asp Met 65 70 75 80 Asp Gly23 252 DNA Mus musculus CDS (1)...(252) 23 aca cct cct tct gac ctg cgcctg agc ccc ctg aca cca tcc acc gtt 48 Thr Pro Pro Ser Asp Leu Arg LeuSer Pro Leu Thr Pro Ser Thr Val 1 5 10 15 cgg tta cac tgg tgt ccc cccacg gag ccc aat ggt gag att gtg gag 96 Arg Leu His Trp Cys Pro Pro ThrGlu Pro Asn Gly Glu Ile Val Glu 20 25 30 tat cta att ctc tac agc aac aaccac acc cag ccc gaa cac cag tgg 144 Tyr Leu Ile Leu Tyr Ser Asn Asn HisThr Gln Pro Glu His Gln Trp 35 40 45 aca ctg ctc acc aca gag gga aac atcttc agt gca gag gtc cat ggc 192 Thr Leu Leu Thr Thr Glu Gly Asn Ile PheSer Ala Glu Val His Gly 50 55 60 cta gag agt gac act cgg tat ttc ttc aagatg gga gcc cgc aca gag 240 Leu Glu Ser Asp Thr Arg Tyr Phe Phe Lys MetGly Ala Arg Thr Glu 65 70 75 80 gtg ggg cct ggg 252 Val Gly Pro Gly 2484 PRT Mus musculus 24 Thr Pro Pro Ser Asp Leu Arg Leu Ser Pro Leu ThrPro Ser Thr Val 1 5 10 15 Arg Leu His Trp Cys Pro Pro Thr Glu Pro AsnGly Glu Ile Val Glu 20 25 30 Tyr Leu Ile Leu Tyr Ser Asn Asn His Thr GlnPro Glu His Gln Trp 35 40 45 Thr Leu Leu Thr Thr Glu Gly Asn Ile Phe SerAla Glu Val His Gly 50 55 60 Leu Glu Ser Asp Thr Arg Tyr Phe Phe Lys MetGly Ala Arg Thr Glu 65 70 75 80 Val Gly Pro Gly 25 26 DNA ArtificialSequence oligonucleotide primer 25 aagcaggtga gcctctctgg cccact 26 26 26DNA Artificial Sequence oligonucleotide primer 26 cttgagacag atccacagctccagac 26 27 30 DNA Artificial Sequence oligonucleotide primer 27atccgggaag ggcttccctg tgggagcttc 30 28 26 DNA Artificial Sequenceoligonucleotide primer 28 gcgctgggga catcgtccag tgtatg 26 29 28 DNAArtificial Sequence oligonucleotide primer 29 gttccaggtc ccgaacctgcagctctgt 28 30 27 DNA Artificial Sequence oligonucleotide primer 30ccactcccct tgccttttgg tagtgaa 27 31 21 DNA Artificial Sequenceoligonucleotide primer 31 gtgctgacct tctgcctgct g 21 32 22 DNAArtificial Sequence oligonucleotide primer 32 ctctgtctgc tacactggtc aa22 33 18 DNA Artificial Sequence oligonucleotide primer 33 tggacgccaaggagttgg 18 34 19 DNA Artificial Sequence oligonucleotide primer 34caaatcccac agaacagga 19 35 17 DNA Artificial Sequence oligonucleotideprimer 35 acgggcatca tcgtggg 17 36 25 DNA Artificial Sequenceoligonucleotide primer 36 gaggaggaca atccgggaag ggctt 25 37 24 DNAArtificial Sequence oligonucleotide primer 37 tcaagcagtt gacacttgac tgtg24 38 24 DNA Artificial Sequence oligonucleotide primer 38 taatctcacagtgatgagag gaga 24 39 26 DNA Artificial Sequence oligonucleotide primer39 ctgtgtctca atcttgaaca aacaca 26 40 25 DNA Artificial Sequenceoligonucleotide primer 40 ggaagagaga cagtaaacat ttcgt 25 41 24 DNAArtificial Sequence oligonucleotide primer 41 ctcccttcct tcctgatcgt tttc24 42 25 DNA Artificial Sequence oligonucleotide primer 42 cggctctcaagcactgcaga ttttg 25 43 500 DNA Mus musculus CDS (276)...(338) 43aggctggtgg cgcgcgggcg cgtgtcccct gtggtgcagg gtggccacac tggcggggcg 60cccccgcgtg ggccgctagc ccaagatggc gatggagggg cgggcgagct ggccgcggcc 120ccggcccccg cgccggcccc cgctcggccc cggccccgga ggcccgcgcc ccgcccgcgg 180cgccgcgcct cccggagcca ctgacgcccg gcgcgccctc ccccggcggc ggcccaggcg 240cccggacgcg gcggcagcgg cccgagcccg gccct atg gcg cgg gcg gac acg 293 MetAla Arg Ala Asp Thr 1 5 ggc cgc ggg ctc ctg gtg ctg acc ttc tgc ctg ctgtcc gcg cgc 338 Gly Arg Gly Leu Leu Val Leu Thr Phe Cys Leu Leu Ser AlaArg 10 15 20 ggtaagggcc cgggtggccg cagtcgcgag tgggcgtccc cggcgcccgcgatgcttgcg 398 cgccgggggc tgtggggact tgcccccagg gggtgtgtgt ccttgctgtgcacagcctgg 458 caccgtgcgt gtccccctgc gcgtggccct tgtgcatgtg ag 500 44 21PRT Mus musculus 44 Met Ala Arg Ala Asp Thr Gly Arg Gly Leu Leu Val LeuThr Phe Cys 1 5 10 15 Leu Leu Ser Ala Arg 20 45 3756 DNA Mus musculus 45atggcgcggg cggacacggg ccgcgggctc ctggtgctga ccttctgcct gctgtccgcg 60cgcggggagc tgccattgcc ccaggagaca actgtcaagc tgagctgtga tgagggaccc 120ctgcaagtga tcctgggccc tgagcaggct gtggtgctgg actgcacttt gggggctaca 180gctgctgggc ctccgaccag ggtgacatgg agcaaggatg gagacactgt actagagcat 240gagaacctgc acctgctacc caatggctcc ctgtggctgt cctcacccct agagcaagaa 300gacagcgatg atgaggaagc tcttaggatc tggaaggtca ctgagggcag ctattcctgt 360ctggcccaca gcccgctagg agtggtggcc agccaggttg ctgtggtcaa gcttgccaca 420ctcgaagact tctctctgca ccccgagtcc cagattgtgg aggagaacgg gacagcacgc 480tttgaatgcc acaccaaggg ccttccagcc cccatcatta cttgggaaaa ggaccaggtg 540accgtgcctg aggagccccg gctcatcact cttcccaagt ggctcctcca gatcctagat 600gtccaggaca gtgatgcagg ctcctaccgc tgcgtggcca ccaattcagc ccgccaacga 660ttcagccagg aggcctcgct cactgtggcc ctcagagggt ctttggaggc taccaggggg 720caggatgtgg tcattgtggc agccccagag aacaccacgg tagtgtctgg acagaatgta 780gtgatggagt gcgtggcctc tgctgacccc accccttttg tgtcctgggt ccgacaggat 840ggaaagccta tctccacgga tgtcatcgtt ctgggccgga ccaatctact catcgccagc 900gcgcagcctc ggcactctgg agtctatgtc tgccgagcca acaagcccct cacgcgtgac 960ttcgccactg cggctgctga gctccgagtg cttgctgccc cagccatctc gcaggcaccc 1020gaggcgctct cgcggacgcg ggccagcacc gcgcgcttcg tgtgccgggc gtccggggag 1080ccacggcccg cgctgcactg gctgcacgac gggatcccgt tgcgacccaa tgggcgcgtc 1140aaggtgcagg gcggtggcgg cagcttggtc atcactcaga tcggcctgca ggacgctggc 1200tactaccagt gcgtagcaga aaacagcgcg ggaactgcct gtgccgctgc gcccctggcg 1260gtagtggtgc gcgaggggct gcccagcgcc ccgactcggg tcacagccac gccgctgagc 1320agctcctctg tgctggtggc ctgggagcgg cctgagttgc acagcgagca aatcattggc 1380ttctctcttc actaccaaaa ggcaagggga gtggacaatg tggagtacca gtttgcagta 1440aacaatgaca ccacagagct gcaggttcgg gacctggaac ccaacacgga ttatgagttc 1500tacgtggtgg cctactccca gctgggggcc agccgaacct ccagcccagc cctggtgcat 1560acactggacg atgtccccag cgcagcaccc cagcttacct tgtccagccc caacccctcg 1620gacatcaggg tggcatggct gcccctgccc tccagcctga gcaatggaca ggtgctgaag 1680tacaagatag agtacggttt ggggaaggaa gatcaggttt tctccaccga ggtgcctgga 1740aatgagacac aacttacgtt aaactcactt cagccaaaca aagtgtaccg agtccggatt 1800tcagctggca ctggcgctgg ctatggagtc ccttctcagt ggatgcagca caggacacct 1860ggtgtgcaca accagagcca tgttcccttt gcccctgcag aattgaaggt gagggcaaag 1920atggagtccc tggtggtgtc atggcagccg ccccctcacc ccacccagat ctctggatac 1980aaactctact ggggagaggt gggaacagag gaggaggcag atggtgaccg ccccccaggg 2040ggtcgtggag atcaagcttg ggacgtcggg cccgtgcggc tgaagaagaa agtgaagcag 2100tatgaactga cccagttagt ccctggcagg ccgtacgagg tgaagctcgt agctttcaac 2160aaacacgagg acggctacgc tgctgtgtgg aagggcaaga cggagaaggc gcccacgcca 2220gacctgccta tccagagggg gccaccgctg cctcctgccc atgtccacgc agagtcaaac 2280agctccactt ccatttggct tcggtggaag aagccagact ttaccactgt caagattgtc 2340aactacactg tacgcttcgg cccctggggg ctcaggaatg cttccctggt cacctactat 2400accagctctg gagaagacat tctcattggc ggcctgaaac catttaccaa gtacgagttt 2460gcggtacagt cccacggagt ggatatggat gggccctttg gctccgtcgt agaacgctcc 2520accctgcctg accggccttc aacacctcct tctgacctgc gcctgagccc cctgacacca 2580tccaccgttc ggttacactg gtgtcccccc acggagccca atggtgagat tgtggagtat 2640ctaattctct acagcaacaa ccacacccag cccgaacacc agtggacact gctcaccaca 2700gagggaaaca tcttcagtgc agaggtccat ggcctagaga gtgacactcg gtatttcttc 2760aagatgggag cccgcacaga ggtggggcct gggccctttt cccgcttgca ggatgtgatt 2820actctgcaag agacattctc agactccttg gatgtgcacg ccgtcacggg catcatcgtg 2880ggtgtctgcc tgggccttct ctgcctcctg gcctgcatgt gtgctggcct acgacaaagc 2940tcccacaggg aagcccttcc cggattgtcc tcctcaggca ccccaggaaa cccagcgctc 3000tacacaagag ctcggcttgg gcctcccagt gtccctgctg cccatgagtt ggagtccctc 3060gtgcatcctc gtccccagga ttggtcccca ccaccctcag atgtggaaga caaggctgaa 3120gtacacagcc ttatgggtgg cagtgtttca gattgccggg gccactccaa gagaaagatc 3180tcctgggctc aggcaggggg accaaactgg gcaggctcct gggcaggctg tgagctgccc 3240cagggtagtg gtccaaggcc ggctctgacc cgtgctctgc tgcctccagc gggaaccggg 3300cagacactgc tgctgcaagc cctggtgtat gacggcataa agagcaacgg gagaaagaag 3360ccgtccccag cctgcaggaa tcaggtggaa gctgaggtca ttgtccactc cgacttcggt 3420gcatccaaag gatgtcctga cctccacctc caagacctgg agccagagga accactgact 3480gcagagactc tgccttccac gtctggagct gtggatctgt ctcaaggagc agactggctg 3540ggcagggagc tgggagggtg ccaaccaaca accagtgggc cagagaggct cacctgcttg 3600ccagaagcag ccagtgcctc ctgctcctgc tcagacctcc agcccagcac tgctatagag 3660gaggcccctg ggaaaagctg ccagcccaaa gccctgtgtc ctctaacagt cagcccaagc 3720cttcccaggg cccctgtctc ctctgctcag gtcccc 3756

I claim:
 1. An isolated Nope polypeptide, or functional fragmentthereof, comprising the amino acid sequence of a Nope polypeptide (SEQID NO:2), or a modification thereof.
 2. The isolated Nope polypeptide ofclaim 1, wherein said functional fragment comprises the amino acidsequence of a Nope polypeptide extracellular domain (SEQ ID NO:4). 3.The isolated Nope polypeptide of claim 2, wherein said functionalfragment comprises an amino acid sequence selected from the groupconsisting of immunoglobulin domain 1 (SEQ ID NO:8), immunoglobulindomain 2 (SEQ ID NO:10), immunoglobulin domain 3 (SEQ ID NO:12),immunoglobulin domain 4 (SEQ ID NO:14), fibronectin domain 1 (SEQ IDNO:16), fibronectin domain 2 (SEQ ID NO:18), fibronectin domain 3 (SEQID NO:20), fibronectin domain 4 (SEQ ID NO:22), and fibronectin domain 5(SEQ ID NO:24).
 4. The isolated Nope polypeptide of claim 1, whereinsaid functional fragment comprises the amino acid sequence of a Nopepolypeptide intracellular domain (SEQ ID NO:6).
 5. An antibody thatspecifically binds the Nope polypeptide of claim
 1. 6. The antibody ofclaim 5, wherein said antibody is a polyclonal antibody.
 7. The antibodyof claim 5, wherein said antibody is a monoclonal antibody.
 8. A methodof detecting a Nope polypeptide, comprising contacting a sample with theantibody of claim 5, and detecting specific binding of said antibody. 9.An isolated nucleic acid molecule encoding a Nope polypeptide amino acidsequence referenced as SEQ ID NO:2, or a modification thereof.
 10. Anisolated nucleic acid molecule comprising the nucleotide sequencereferenced as SEQ ID NO:1, or a modification thereof.
 11. The nucleicacid molecule of claim 10, wherein said nucleotide sequence is selectedfrom the group consisting of SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19,21 and
 23. 12. A Nope oligonucleotide, comprising between 15 and 300contiguous nucleotides of SEQ ID NO:1 or the anti-sense strand thereof.13. The isolated Nope oligonucleotide of claim 12, wherein saidoligonucleotide comprises between 15 and 300 contiguous nucleotides ofSEQ ID NO:5 or the anti-sense strand thereof.
 14. A vector comprising anexpression element operationally linked to the nucleotide sequence ofclaim
 10. 15. A host cell comprising the vector of claim
 13. 16. Amethod of detecting a Nope nucleic acid molecule in a sample, comprisingcontacting said sample with a Nope oligonucleotide of claim 12 underconditions allowing specific hybridization to a Nope nucleic acidmolecule, and detecting said specific hybridization.
 17. A method ofdetecting a Nope nucleic acid molecule in a sample, comprisingcontacting said sample with a Nope oligonucleotide of claim 13 underconditions allowing specific hybridization to a Nope nucleic acidmolecule, and detecting said specific hybridization.
 18. A method ofdetecting a Nope nucleic acid molecule in a sample, comprisingcontacting said sample with two or more Nope oligonucleotides of claim12, amplifying a nucleic acid molecule, and detecting saidamplification.
 19. The method of claim 18, wherein said amplification isperformed using polymerase chain reaction.
 20. A kit comprising one ormore Nope oligonucleotides comprising between 15 and 300 contiguousnucleotides of SEQ ID NO:1 or the anti-sense strand thereof.